The conformation of vitronectin (Vn) in solution and adsorbed to hydrophilic and hydrophobic glass was studied using the polarity-sensitive fluor acrylodan. Free sulfhydryl groups of vitronectin were labeled with acrylodan under nondenaturing conditions. The number and location of the free sulfhydryl groups were determined by cleavage with either formic acid or cyanogen bromide followed by polyacrylamide gel electrophoresis. Based upon published amino acid sequences of Vn, the fluorescence of acrylodan-labeled Vn (Ac-Vn) fragments is consistent with free sulfhydryls being located at cysteine residues 196 and 274. Conformational changes upon adsorption on glass and silanized glass surfaces placed the free sulfhydryl residues in a more hydrophobic environment than was observed when Vn was in PBS solution. Small but statistically significant quenching of Ac-Vn fluorescence with iodide could be explained in terms of a two-site model. Results of this study suggest that the domains surrounding the free sulfhydryl groups of vitronectin experience different environments in solution and adsorbed to surfaces.