Tightly confluent monkey cell lines BSC-1 and CV-1 held in stale medium for several days exhibited an extremely low level of thymidine incorporation into cellular DNA. Yet, these cells contained a level of alpha-polymerase equal to about 15% of the level in rapidly dividing cells, and they still were capable of supporting replication of SV40 DNA. SV40 infection and culture in stale medium resulted in a four-fold induction of alpha-polymerase in CV-1 cells, whereas no change in alpha-polymerase level was observed in BSC-1 cells. Characterization of alpha-polymerase partially purified from infected CV-1 cells revealed that 80-90% of the enzyme activity was aphidicolin resistant. SV40 DNA replication in resting CV-1 cells, however, was aphidicolin sensitive. SV40 infection of resting CV-1 cells may induce an aphidicolin-resistant enzyme or lead to a modified alpha-polymerase species.