Previous analyses have suggested that the CD45 tyrosine phosphatase activates src family tyrosine kinases p56lck and p59fyn by dephosphorylating regulatory COOH-terminal residues. We have examined the status of p56lck and p59fyn in murine and human CD45- T cell lines. Surprisingly, despite the fact that p56lck and p59fyn were spontaneously hyperphosphorylated, the tyrosine kinase activity of both enzymes was increased in CD45- versus CD45+ cells. In vitro exposure of hyperphosphorylated p56lck to CD45 decreased enzyme activity to near-basal levels. Lck from CD45- cells was hyperphosphorylated on the cyanogen bromide digestion fragment that contains the negative regulatory residue Tyr-505, and the identity of this site of phosphorylation was confirmed by trypsin digestion followed by high performance liquid chromatography. Loss of CD45 results, therefore, in a paradoxical hyperphosphorylation of the COOH-terminal tyrosine and increased src family kinase enzymatic activity.