An antibody (Ab) can be specifically labeled with fluorophores in the vicinity of its antigen-combining sites by using photoaffinity labeling techniques. Iodide quenching df such fluorophores was used to determine the orientation of the Ab on dichlorodimethylsilane (DDS)-treated silica surfaces. A control experiment was also performed using Abs labeled in the hinge region with the same fluorophore. Two different fluorescently-labeled Abs were investigated-polyclonal goat anti-biotin Abs conjugated with fluorescein maleimide and monoclonal anti-fluorescein Ab (9-40) conjugated with tetramethylrhodamine maleimide. The fluorescent emission of labeled Abs adsorbed on hydrophobic DDS-silica surfaces was excited by the evanescent wave generated by total internal reflection techniques. Ioide quenching data were analyzed using a modified Stern-Volmer equation, which assumes both accessible and inaccessible populations of fluorophores. The experimental results showed that the antigen-binding fragments (Fab) of acid-pretreated Abs adsorbed to DDS - silica surfaces were more exposed to buffer solution than their constant fragments. Acid pretreatment of an Ab leads to increased antigen-binding capacity on DDS surfaces due to the increased accessibility of Fab regions to buffer solution.