Chinese hamster kidney cells are semi-permissive to simian virus 40 (SV40). Exposure to mitomycin C (MC) of Chinese hamster kidney cells infected with SV40 DNA enhanced the yield of infectious virus 10- to 100-fold. This stimulation occurred whether the treatment was performed before or after infection. A simultaneous increase in the number of V antigen-synthesizing cells and virus-producing cells, as well as the virus burst size, was observed upon MC pretreatment, whereas the proportion of T antigen-synthesizing cells remained unchanged. MC pretreatment clearly stimulated virus DNA replication in SV40 virus-infected cells. Cells treated with MC exhibited an unbalanced growth pattern, with continuing protein synthesis in the absence of cell division and a markedly reduced ability to replicate the cellular DNA. These results suggest that MC enhances the permissiveness of Chinese hamster kidney cells by inducing the synthesis of a specific cellular factor(s) required for SV40 reproduction in Chinese hamster kidney cells.