The nasal mucosa provides a non-invasive route for drug administration to the systemic circulation and potentially directly to the CNS. Nanoparticles made from biodegradable polymers, including PLGA, are of great interest for use in drug delivery systems due to their relative safety and ease of surface modification. Owing to their small size, nanoparticles may provide enhanced targeting and transport through the nasal mucosa. An improved understanding of the mechanisms and pathways of nanoparticle transfer across the nasal mucosa is needed to design effective new nasal delivery systems. This study focuses on the preparation of PLGA nanoparticles in various diameters and with varying surface characteristics followed by the in vitro investigation of the mechanisms of endocytosis and exocytosis of PLGA nanoparticles in the nasal mucosa. PLGA nanoparticles (60 nm or 125 nm) containing the lipophilic fluorescent dye, Nile Red, were prepared using a surfactant-free nanoprecipitation method. In one investigation, the inherent negative surface charge of 60 nm PLGA nanoparticles was modified to a positive charge using a 5th generation polyamidoamine dendrimer (PAMAM) during preparation of nanoparticles. In addition, 60 nm PLGA nanoparticle surfaces were coated by adding 5 % (w/v) bovine serum albumin (BSA) to the nanoparticle dispersion and allowing protein adsorption on the particle surface. Nile Red-loaded PLGA nanoparticles were transported into the epithelial layer and reached the sub-mucosal connective tissues, yet only < 5% of the PLGA nanoparticle load was transferred into the nasal mucosa. Total uptake was size dependent, where the uptake of 60 nm unmodified PLGA nanoparticles was significantly higher than uptake of 125 nm nanoparticles. The amount of Nile Red measured in the tissues after expose to the 125 nm nanoparticles was double the amount from the 60 nm nanoparticles due to differences in the carrying capabilities of the 60 and 125 nm PLGA nanoparticles. Modification of the nanoparticle surface with PAMAM or BSA decreased the uptake of 60 nm PLGA nanoparticles into the nasal mucosa. Endocytic mechanisms involved in the uptake of PLGA nanoparticles were studied using chemical inhibitors. Nanoparticle uptake in the nasal respiratory mucosa involved energy-dependent processes utilizing multiple known mechanisms, including clathrin-mediated endocytosis and macropinocytosis. In the olfactory mucosa, significant energy-independent nanoparticle uptake was also observed. In order to investigate how nanoparticles exit epithelial cells for further distribution to distant tissues, the exocytosis of 60 nm Nile Red-loaded PLGA nanoparticles was evaluated using three different epithelial cell line models, RPMI-2650 (nasal), Calu-3 (lung) and MDCK-II wild type (kidney) cells. Following a 30 min exposure to a 60 nm PLGA nanoparticles dispersion, nanoparticle exocytosis into a protein-free medium was evaluated for additional 30 or 60 min. Only a limited number of NP (~ 20 % of the endocytosed NP) underwent exocytosis into the medium after 60 min, while the majority of the internalized nanoparticles remained within the cells. The measurable transfer of PLGA nanoparticles into the nasal mucosal tissues indicates that they may be useful drug carriers for nasal administration. However, the limited exocytosis of 60 nm NP and the resulting potential for intracellular accumulation may raise toxicity concerns and result in potential cellular injury. While PLGA nanoparticles provide promising drug delivery systems for nasal administration, only with careful design of the nanoparticles, including their size and surface characteristics, will efficient and effective, safe drug delivery be accomplished.