We have found in different human cells of lymphoid and non-lymphoid origin that the 56 kilodalton (kDa) lck protein is rapidly converted to a product migrating at approximately 60 kDa (designated p60lck) in response to the phorbol ester 4 alpha-phorbol 12 beta-myristate (PMA) as well as the diacylglycerol analogue 1,2-dioctanoyl-sn-glycerol (diC8). This conversion is associated with an increase in serine phosphorylation within the amino-terminal 18 kDa portion of the lck protein. The serine phosphorylation modification and diminished electrophoretic mobility of the lck protein appear to be completely reversible within 60 min following treatment with diC8. The changes in p56lck phosphorylation and gel mobility in response to activators of protein kinase C are also associated with a small but reproducible decrease in the ability of the lck protein to be phosphorylated in immune complex kinase assays. While these alterations of the lck gene product may play an important role in antigen-mediated activation of T-lymphocytes, we demonstrated that they can also be induced independently of T-cell activation suggesting that they are not necessarily implicative of this process.