Potent drugs such as cyclosporine have provided effective probes of signal transduction pathways and, as well, of human immunodeficiency virus type 1 (HIV-1) replication mechanisms. Recently, it was reported that As(2)O(3), a drug used to treat acute promyelocytic leukemia (PML), stimulates HIV-1 replication. We found that As(2)O(3) accelerates the kinetics of a spreading HIV-1 infection in human T cells and increases the number of cells bearing HIV-1 provirus after a single round of infection. The stimulatory effect occurred after membrane fusion and resulted in increased steady-state levels of newly synthesized viral cDNA. Stimulation was independent of HIV-1 env and most viral accessory genes, and As(2)O(3) had no detectable effects on viral expression postintegration or virion assembly. Murine leukemia virus (MLV) transduction was enhanced by As(2)O(3) to the same extent as HIV-1 transduction, but As(2)O(3) had no additional effect on Fv1 restriction. In contrast, As(2)O(3) largely overcame the specific block to N-tropic MLV reverse transcription posed by human Ref1. As(2)O(3) disrupts PML bodies, nuclear structures named for a major component, the PML protein. We observed no changes in PML bodies in response to HIV-1 infection. Experiments with PML-null target cells indicated that PML has no effect on HIV-1 infectivity and is dispensable for the stimulatory effect of As(2)O(3). As(2)O(3) caused cell death in uninfected cells at the same concentrations which stimulate HIV-1 replication. Among four additional apoptosis-inducing agents, a boost in HIV-1 infectivity was observed only with carbonyl cyanide m-chlorophenylhydrazone, a compound which, like As(2)O(3), disrupts the mitochondrial transmembrane potential. In summary, As(2)O(3) stimulates retroviral reverse transcription, perhaps via effects on mitochondria, and provides a useful tool for characterizing Ref1.