Lymphocyte membrane fractions from both normal and neoplastic sources exhibit tyrosine-specific protein kinase activity. The molecular weights of the endogenous substrates phosphorylated on tyrosine residues differ in B and T cells. To further characterize membrane tyrosine phosphorylation in the two major classes of lymphocytes, the tryptic phosphopeptides of their endogenous substrates were compared and the sensitivity of the kinases to inhibition by N(α)-p-tosyl-L-lysine chloromethyl ketone (TLCK) was determined. The two major B cell substrates (61,000 and 55,000 daltons, p61 and p55) were gel purified after phosphorylation and exhaustively digested with trypsin. Separation by reverse phase high pressure liquid chromatography demonstrated that these two substrates had two identical phosphotyrosine containing tryptic phosphopeptides. p61 had an additional phosphotyrosine site. Parallel analysis of the two T cell substrates (64,000 and 58,000 daltons, p64 and p58) showed that they also contained two phosphotyrosine sites that were identical. However, the tryptic phosphopeptides from the B and T cell substrate pairs were clearly distinct suggesting that they arise from different gene products. When B and T cell membrane fractions were preincubated with TLCK (21 °C, 30 min) a dose-dependent decrease in p64 and p58 phosphorylation resulted. p61 and p55 phosphorylation was not affected at concentrations up to 10 mM TLCK. Tyrosine-specific kinase activity was also assessed by measuring phosphorylation of a tyrosine containing synthetic peptide. The kinase activity of T cell plasma membrane fractions was inhibited by TLCK; the B cell activity was unaffected. The results suggest that membrane fractions from normal and some neoplastic B and T cells have at least two different tyrosine-specific kinases.