Simian virus 40 (SV40) infection of quiescent monkey kidney cells stimulates two successive rounds of cellular DNA synthesis without an intervening mitosis. This uncoupling of S phase and mitosis indicates that SV40 modulates pathways regulating the G2-to-M phase transition. To examine the integrity of mitotic initiation pathways in infected cells that have bypassed mitosis, SV40-infected CV-1 cells were treated with okadaic acid (OA), a known inducer of premature mitosis in other cell types. OA treatment triggered the appearance of the mitotic marker MPM-2 in SV40-infected CV-1 cells progressing through either the first (diploid) or second (tetraploid) S phases. These results demonstrate that a subset of mitotic pathways are intact but inactive in SV40-infected cells that have bypassed mitosis and initiated tetraploid S phase.