Macrophages are uniquely responsive to bacterial lipopolysaccharide (LPS) for activation of a number of host defense functions and production of bioactive mediators. One potentially important mediator produced by LPS-stimulated macrophages is interferon (IFN-α/β). In contrast to murine observations, we have observed that freshly isolated human monocytes, purified by counter-current centrifugal elutriation, do not produce interferon in response to LPS. This is not due to a lack of response to LPS, as assessed by the induction of other monokines, or to an incapacity for IFN production, since IFN was inducible by poly-l,C treatment of monocytes in the absence of any other exogenous stimulus. However, human monocytes can be primed for the production of IFN in response to LPS if they are cultured in the presence of either granulocyte-macrophage colony stimulating factor (GM-CSF) or interferon-γ (IFN-γ). The IFN secreted is of the α subtype. Monocytes primed with GM-CSF or IFN-γ also maintained LPS responses for production of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1). M-CSF did not prime monocytes for LPS-induced IFN production, although it did enhance production of TNF-α and promoted monocyte survival. Northern analysis indicated that the induction of IFN-α by LPS was regulated primarily at the mRNA level. The highly regulated production of IFN-α by monocytes/macrophages has important implications for autocrine action of interferons in the activation and differentiation of these cells.