Human immunodeficiency virus type 1 (HIV-1) infection of the CD4+ SupT and CEM cell lines, blocked in cell replication by the polymerase alpha inhibitor aphidicolin (APC), was studied. The APC-treated cells showed a lack of viral production, but the presence of single cell killing. High levels of unintegrated viral DNA forms were found in the infected APC-treated cells as compared with untreated cells. Moreover, an increased rate of viral replication occurred in the remaining viable cells following removal of APC. The results indicate that HIV-1 entry and reverse transcription can take place in cells blocked in the S phase of the cell cycle. Replication of infectious progeny virions appears to require de novo cell division. Finally, accumulation of viral DNA in cells during APC treatment can result in cytopathological effects and subsequent enhancement of virus production.