p56lck, a member of the src family of non-receptor protein tyrosine kinases, is expressed almost exclusively in cells of lymphoid origin. Recent evidence has implicated p56lck in a critical role both in T cell development and activation. A variety of T cell stimuli induce a shift in the electrophoretic mobility of p56lck from an apparent molecular mass of 56 kDa (p56lck) to 60 kDa (p60lck). This shift in electrophoretic mobility correlates with an increase in the phosphoserine content of the p60lck. We have shown that both 4 alpha-phorbol 12 beta-myristate acetate and OKT3 treatment of Jurkat cells, as well as 4 alpha-phorbol 12 beta-myristate acetate treatment of 171.CD4 and LSTRA cells, induced phosphorylation of serine-59 on p56lck in vivo, which correlated with the shift to p60lck. We also demonstrated that the same serine residue could be phosphorylated in vitro with mitogen-activated protein kinases and that this event was capable of reducing p56lck activity in vitro. Combined, these data suggest a novel pathway for the in vivo regulation of p56lck activity.