p34cdc2 kinase, an enzyme essential for mitosis in mammalian cells, may play a role in etoposide-induced G2 phase arrest of Chinese hamster ovary cells. In this study, etoposide is shown to cause inhibition of a specific p34cdc2 kinase activation pathway, that of tyrosine dephosphorylation. Exposure of asynchronously dividing cells to etoposide caused a simultaneous rapid decline of both mitotic index and p34cdc2 kinase activity, suggesting that the kinase was not activated and that the arrest point was in late G2 phase. Using synchronized cells, p34cdc2 kinase exhibited maximal activity at the G2/M transition. Activation of the kinase and the onset of mitosis were accompanied by increased electrophoretic mobility and tyrosine dephosphorylation of the p34cdc2 protein. A 1-h exposure to etoposide during early G2 phase inhibited p34cdc2 kinase activation, its shift in electrophoretic mobility, and its tyrosine dephosphorylation, all of which correlated with a delay in mitotic progression. The interaction between the p34cdc2 and cyclin B proteins appeared unaffected under etoposide exposure conditions which resulted in greater than 70% inhibition of p34cdc2 kinase activity and almost complete cessation of transition into mitosis. These data suggest that mammalian cells express a DNA damage-responsive mechanism which controls mitotic progression at the level of p34cdc2 tyrosine dephosphorylation.