The replicative DNA polymerase alpha is an intracellular target of aphidicolin. In vitro this drug inhibits DNA polymerase alpha reversibly. Yet, its in vivo effect on SV40 DNA replication, which depends on DNA polymerase alpha, was found to be irreversible. Thus, exposure of infected cells to aphidicolin led to a progressive loss in their ability to incorporate [3H]dT into viral DNA in a subsequent pulse without drug. This loss was time-dependent (t1/2 at 37 degrees C at 2 microgram/ml of drug was approximately 20 min) and increased with drug concentration. Likewise, replicating SV40 DNA, pulse-labeled prior to exposure, lost the ability to mature into form I DNA upon removal of the drug. No degradation of replicating SV40 DNA molecules was detected by neutral sucrose gradient analysis during or up to 1 h after aphidicolin exposure. However, longer incubations resulted in breakdown of the arrested replicative intermediate, concomitant with the resumption of viral DNA synthesis. Origin-synchronized SV40 replicons were less affected by exposure to aphidicolin than were ongoing replicons, as judged from comparing recoveries of tsA replicons from 40 degrees C restriction, with or without the drug. The data indicate that replicating SV40 chromosomes become selectively impaired during aphidicolin arrest and prevent thereby the initiation of new replication rounds, perhaps by occupying fixed nuclear replication sites.