Membrane fractions isolated fom mouse and rat spleen expressed substantial tyrosine-specific protein kinase activity. Phosphotyrosine (P-Tyr) accumulation in endogenous membrane substrates was stimulated by vanadate or nonionic detergents. When in vitro phosphorylation was carried out at 0°C in the presence of 1 mM Mn2+ and Triton X-100, P-Tyr constituted up to 40-50% of the total phospho amino acid. Polyacrylamide gel electrophoresis showed that membranes from mixed lymphocyte populations have four major P-Tyr-containing proteins. Whereas nonionic detergents were potent stimuli for P-Tyr accumulation in all four substrates, tyrosine phosphorylation of two of these (p61 and p55) was markedly dependent on vanadate. These two substrates were present in membranes from surface Ig-bearing splenic lymphocytes purified by affinity chromatography and Raji, a human B-lymphoblastoid cell line. P-Tyr accumulation in the two other substrates observed in splenocyte membranes (p64 and p58) was much less dependent on vanadate. p64 and p58 were phosphorylated in membranes from mouse thymocytes and human and mouse T-lymphoma cell lines, while p61 and p55 were not. Thus it appears that in both murine and human lymphocytes, p64 and p58 served as T-cell-specific substrates, while p61 and p55 were specifically associated with B lymphocytes. Moreover, these distinct P-Tyr substrate patterns were conserved in some neoplastic cell lines derived from B and T cells.