Nutrient limitation in aquatic ecosystems results from a deficiency in nitrogen or phosphorus levels relative to cellular growth needs. Nutrient limitation of freshwater systems is a function of biotic and abiotic factors. Biotic factors include vascular and nonvascular plant community composition. Abiotic factors include underlying bedrock and land-use activities (e.g. agriculture, septic systems). Nutrient availability directly affects growth, productivity, and community structure of primary producers. The purpose of this study was two-fold: (1) to assess the relationship between ambient algal biomass. and in-stream nutrient levels along the longitudinal course of a river through a transition from weak to well-developed underlying karst bedrock, and (2) experimentally assess if periphyton was nitrogen or phosphorous limited between weak and well-developed karst sites. Sestonic and filamentous biomass (= chlorophyll-a) levels increased monthly along the longitudinal gradient. In contrast, periphyton biomass levels increased minimally monthly and displayed no longitudinal pattern. Nitrate and soluble reactive phosphorus levels exhibited distinct longitudinal increases, whereas total phosphorous displayed minimal change and ammonia levels decreased in the downstream direction. Total nitrogen (TN) levels increased upstream but decreased sharply in the well-developed downstream karst sites. The nutrient limitation assays revealed that the highest periphyton levels were with N + P treatments at the most upstream sites. Overall, in Kentucky's Green River algal biomass accrual appears to be mainly P-limited but likely also by TN availability during late summer.