Barnacles are dominant hard–fouling organisms in marine waters. They attach to substrates by secreting a complex proteinaceous adhesive. Understanding the chemical composition of this multi–protein underwater adhesive and how it is affected by environmental variables, such as oceanic temperatures, is critical for developing nontoxic solutions to control biofouling. Previous experiments in our lab revealed an inverse relationship between critical removal stress (CRS) and temperatures at which barnacles were reared. Further investigations showed that this correlation is not attributed to differences in physical properties such as barnacle size or short–term changes in the viscosity of adhesive. Therefore, the observed effects may be influenced by a physiological response to temperature during initial growth and development. We hypothesized that rearing temperature affects the expression of proteins found in the adhesive matrix. To elucidate the underlying mechanisms responsible for the temperature effect, we analyzed uncured barnacle adhesive using two-dimensional gel electrophoresis (2DGE) and matrix-assisted laser desorption/ionization-tandem time-of-flight (MALDI-TOF/TOF) mass spectrometry (MS). In our analysis, we 1) detected differences in protein expression at two experimental temperatures (15°C and 25°C) and 2) identified several proteins that may serve functional roles in the process of adhesion. Our data are also consistent with a model that the curing process of barnacle adhesive may be analogous to the process of wound healing in animals.