The kinase activity of the proto-oncogene product, c-Src, increases during mitosis through partial dephosphorylation of Tyr527, the negative regulatory site of c-Src. To examine whether or not Csk, a candidate kinase specific for Tyr527, is involved in this regulation, we developed a Balb/c 3T3 cell line overexpressing Csk and a Csk-deficient cell line. The overexpression of wild-type Csk caused significant suppression of the c-Src activity during mitosis. A membrane-targeted Csk, which has an amino-terminal myristylation signal of c-Src, exhibited an effective suppression of the c-Src activity, even though its expression level was lower than that of endogenous Csk. Concomitant with the suppression of the c-Src activation, the level of tyrosine phosphorylation of a cortactin-related protein, a potential substrate of c-Src in vivo, was reduced. In contrast, the Csk-deficient cells exhibited constitutive activation of c-Src, which showed no significant change in its activity during mitosis. These results suggest that Csk indeed participates in the regulation of the c-Src activity during mitosis.