Several different nucleoside analogs have been demonstrated to inhibit retroviral RNA-dependent DNA polymerase activity in preference to cellular DNA-dependent DNA polymerases. 3′-Amino derivatives of 3-deoxyadenosine was analyzed for their antiviral activity toward HIV-1 and for their host cell toxicity. Puromycin aminonucleoside (PANS), PANS 5′-monophosphate, and 3′-amino-3′-deoxyadenosine triphosphate all inhibited HIV-1 replication in acutely infected cells. No significant antiviral effects of PANS were demonstrated in chronically infected cells. The effect of PANS was demonstrated at an early step in HIV-1 replication, most likely reverse transcription. 3′-Aminonucleoside analogs are a novel class of inhibitors of HIV-1 replication that require further analysis in cell culture and animal studies.