An antigen binding assay has been developed for quantitation by flow cytometry of vesicular and soluble Ia binding by alloactivated T cells. Binding of stimulator membrane vesicles was detected by anti-Ly-6.2 or anti-Ia monoclonal antibodies coupled to fluorescent latex beads. Vesicle binding by an I-Ak specific A.TH anti-A.TL T cell line occurred via I-Ak molecules, in that (a) vesicles expressing I-Ak molecules bound much more effectively than vesicles of H-2b,q strains, and (b) inhibition of H-2k vesicle binding occurred with anti-I-Ak, but not anti-Kk, anti-Ek, or anti-Dk antibodies. T cell receptor/Ia interactions were directly studied by inhibition of H-2k vesicle binding by T cells with partially purified Ia glycoproteins. Inhibition of binding occurred via Ia molecules since (a) affinity column partially purified allogeneic I-Ak molecules inhibited binding much more effectively than syngeneic I-As molecules and (b) depletion of I-Ak but not Ek molecules in Iak containing glycoprotein fractions abrogated the inhibitory effect. The ability of this method to detect specific binding of soluble Ia with antigen activated T cells makes it a useful tool for studying interaction of membrane free major histocompatibility complex (MHC) products with native T cell receptor.