Interferons (IFNs) inhibit the replication of a wide range of animal viruses by acting at various steps of the life cycle. Interferons display a particularly potent antiviral effect on HIV-1 replication in primary human macrophages. A high virus-to-cell multiplicity of infection was used to investigate which steps in a single replicative cycle in these primary human cells were affected by IFNs. Monocyte-derived macrophages from healthy seronegative donors were infected with HIV-1BaL. Virus production was assessed by immunoassay for p24 antigen. Viral DNA was detected by PCR while mRNA was detected specifically by RT-PCR with primers bracketing the 5' introns of HIV-1 to detect only spliced transcripts such as tat, rev, nef, and env mRNAs. Macrophages pretreated with IFN-alpha, -beta or -gamma had a reduced viral DNA signal while the spliced mRNA signal was essentially abolished. No virus was produced. To test whether IFNs could reduce HIV transcripts in cells with established productive infection, macrophages were infected and reinfection was then prevented by azidothymidine before starting interferon treatment. Under such conditions, the addition of interferons did not affect significantly the levels of HIV spliced transcripts. No intracellular accumulation of p24 antigen was observed. Therefore, the major effect of IFNs was at an early step of the virus life cycle and resulted in a reduced viral DNA synthesis.