Antigen-binding inhibition studies using microscopic autoradiography were performed on T or B cell-enriched lymphocyte populations. Antibodies specific for the "framework" of immunoglobulin heavy or light chain variable domains (VH or VL), or anti-H-2 and anti-Ia antisera were used. T cell subclasses were separated with anti-Lyt antisera and complement. It was found that antigen-binding T cells of different subclasses can be inhibited selectively with only one of the two anti-V region antibodies. Antigen binding to Lyt-1+ cells was inhibited by anti-VH, while Lyt-2+,3+ cells were inhibited by anti-VL specifically. Anti-Ia antisera inhibited unprimed Lyt-1+ antigen-binding cells, whereas anti-H-2K or anti-H-2D anti-sera inhibited unprimed Lyt-2+,3+ antigen-binding cells, and both classes of immune T antigen-binding cells.