The new antiviral agent, azidothymidine (AZT; BW A509U), is currently the only successful drug in use for patients with acquired immunodeficiency syndrome. The effect of this thymidine analog, 3'-azido-3'-deoxythymidine, on the replication of the lymphadenopathy-associated virus strain of the human immunodeficiency virus was evaluated by using susceptible H9 and Jurkat cells. Cells were pretreated with concentrations of drug ranging from 0.5 to 100 microM, infected, and maintained in medium containing drug. Virus production was assayed by reverse transcriptase assays, and virus-specific DNA was analyzed by Southern blots probed with cloned human immunodeficiency virus sequences. At 4 to 8 days postinfection, infected cells without drug reached a peak of reverse transcriptase activity that was sustained. Increasing concentrations of AZT caused increasing delays in virus production; however, replicate cultures at nontoxic levels of the drug (up to 25 microM) eventually produced as much virus as did non-drug-treated infected cells, despite the continued presence of the drug. Levels of intracellular, unintegrated, virus-specific DNA paralleled reverse transcriptase levels. Virus-caused cytopathic effect was likewise delayed in drug-treated cultures. Virus recovered from H9 cultures after 25 microM AZT treatment did not appear resistant to AZT.