The order of appearance of human immunodeficiency virus type 1 (HIV-1) nucleic acids was examined in monocyte-derived macrophages following a high multiplicity infection with macrophage-tropic virus. Using the polymerase chain reaction, viral DNA was first detected 2 h after infection and continued to accumulate over the next 24 h. Transcripts representing tat, rev and nef splicing were detected by 24 h, and transcripts representing env splicing were detected by 48 h after infection. Coincident with the appearance of env transcripts, new synthesis of cellular and extracellular p24 antigen began, multinucleated giant cells formed and progeny infectious virus emerged. This analytical system provides a foundation for further studies on the effects of antiviral agents and cellular factors on the replication cycle of HIV-1 in non-transformed, primary monocyte-derived macrophages.