The mechanism of inhibition of HIVBa-L replication by interleukin 10 (IL-10) in primary monocytes and macrophages at various stages of maturation was investigated using semiquantitative PCR for reverse-transcribed HIV DNA, and Northern hybridization for HIV mRNA expression in comparison with extracellular p24 antigen. Pretreatment of monocytes with IL-10 markedly inhibited expression of both unspliced and spliced HIV RNA, reaching a nadir at 7 days and recovering to normal levels by 10 days after a single application. The optimum inhibitory concentration was 25 ng/ml. Less inhibition of HIV RNA expression was observed when IL-10 was added after HIV infection of monocytes and the inhibitory effect progressively declined to negligible levels as monocytes matured into macrophages over 10 days. IL-10 also downregulated the expression of cellular genes, including the transferrin receptor, 28S rRNA, and GAPDH. The kinetics of the inhibition of cellular mRNAs correlated with the inhibition of HIV RNA and also declined as monocytes matured into macrophages. In contrast, IL-10 did not inhibit cellular mRNA expression in the macrophage cell line THP-1. Neutralizing polyclonal antibody to IL-10 reversed all its inhibitory effects. Interaction of IL-10 and TNF-alpha in combination were generally antagonistic in their effects on HIV transcription. IL-10 prevented stimulation of HIV RNA expression by TNF-alpha after preincubation with monocytes for 48 hr. IL-10 had no effect on the levels of HIV cDNA or the process of initiation and completion of reverse transcription. The inhibitory effect of IL-10 on HIV replication in maturing monocytes was probably mediated mainly by inhibition of cellular gene expression and inhibition of maturation of monocytes into macrophages and their activation, with consequent downregulation of HIV mRNA.