Interleukin-12 (IL-12) is a monocyte/macrophage-derived cytokine that is critical for T lymphocyte and natural killer cell activities and functions. In this study, we examined the regulation of IL-12 expression by human monocytes in response to bacterial lipopolysaccharide (LPS). Several novel aspects of IL-12 induction from monocytes were shown. Optimal expression of IL-12 mRNA and bioactivity required specific priming of monocytes by interferon-gamma (IFN-gamma) before LPS stimulation. Granulocyte-macrophage colony-stimulating factor (GM-CSF) provided an equivalent priming stimulus for LPS-induced tumor necrosis factor (TNF) and IL-12 p40 mRNA, but primed poorly for LPS-inducible p35 message and secreted IL-12 activity. Macrophage colony-stimulating factor (M-CSF), although a potent survival factor for monocytes, showed no priming activity for IL-12 production. Time course experiments demonstrated independent regulation of p40 and p35 by IFN-gamma and LPS. LPS inducibility of p40 expression required only a brief exposure to IFN-gamma (2 hours), while prolonged exposure (+/- 24 hours) to IFN-gamma resulted in diminishing levels of p40 mRNA. p35 inducibility (by LPS) required a longer exposure to IFN-gamma (8 to 16 hours), and continued to be inducible up to 40 hours following IFN- gamma priming. Both mRNAs were rapidly induced (1 to 2 hours) in IFN- gamma-primed monocytes; p35 message reached a plateau by 2 hours, while p40 continued to accumulate. Finally, both p40 and p35 were directly induced by LPS in the presence of cycloheximide. These results indicated that both p40 and p35 are LPS-inducible in monocytes following IFN-gamma pretreatment, and that the regulated expression of p35 controls the level of active IL-12 protein in purified human monocytes. The selectivity of priming by IFN-gamma is in accord with a putative role for IL-12 in the initiation and amplification of TH1-type responses.