To facilitate the identification of phosphotyrosine (Ptyrkcontaining proteins, rabbit polyclonal antibodies and mouse monoclonal antibody specifically reactive to P-tyr were prepared by hyperimmunizing the animals with Ptyr-conjugated bovine serum albumin or poly-L-lysine. AS determined by a solid-phase radioimmunoassay and an enzyme-linked immunosorbent assay, the antibodies reacted with P-tyr-conjugated target antigens but not with those conjugated with phosphoserine (P-ser) or phosphothreonine (P-thr). This immune reaction was strongly blocked by 2 mM P-tyr and phenylphosphate but not by Pser or P-thr. The antibodies were capable of isolating, as the major P-tyr-containing components, a I70kd protein (most likely the EGF receptor) from EGF-stimulated, "Plabelled A43 I cells, and l30kd and 60kd proteins from b u s sarcoma virus (RSV)-transformed chick cell lysate which had been labelled in vitro with Y-'~P-ATP. Immunofluorescent staining of RSV-transformed cells and A43 I cells showed specific localization of P-tyr-containing proteins in the cytoplasm, plasma membrane, and nucleoluslike structures. The results demonstrated the usefulness of the antibodies for identification or isolation of P-tyr-containing proteins.