pp60c-src, a cellular tyrosine kinase homologous to the retroviral v-src oncogene, becomes transiently activated during mitosis. Activation is accompanied by phosphorylation of three sites in the amino-terminal regulatory domain of the protein, threonine 34, threonine 46 and serine 72. These sites can be phosphorylated in vitro by a cell cycle-regulated kinase, p34cdc2, yet this does not result in increased kinase activity of pp60c-src. pp60c-src is negatively regulated by phosphorylation at tyrosine 527, and it has been shown that this site is transiently dephosphorylated in mitotic cells. The importance of tyrosine 527 in the regulation of pp60c-src is also emphasized by the fact that oncogenic mutants of pp60src lacking tyrosine 527 are constitutively active during the entire cell cycle. Here we report that a non-myristylated mutant of pp60c-src is not activated and only partially phosphorylated at the amino terminus in mitotic cells. Additional mutants lacking one (TTAc-src), two (AASc-src) and three (AAAc-src) cdc2 phosphorylation sites had slightly higher kinase activity than wild-type pp60c-src in interphase cells and were not activated during mitosis. However, all four mutant proteins were still transiently dephosphorylated at tyrosine 527 during mitosis, suggesting that myristylation and amino-terminal phosphorylation may be necessary but are clearly not sufficient for mitosis-specific activation.