We have evaluated the potential role of prostaglandins and their second-messenger Cyclic Adenosine Monophosphate (cAMP) in the activation of interleukin-6 (IL-6) promoter regulatory elements leading to IL-6 expression in monocytic cells. We demonstrate that prostaglandins of the E series and their second-messenger cAMP induce the IL-6 promoter in the murine monocytic cell line PU5-1.8. Stimulation with both cAMP and LPS results in a marked synergistic effect. We show that the endogenous IL-6 gene is induced by cAMP as well, even though to a lesser extent than by LPS, suggesting distinctive effects of cAMP and LPS on posttranscriptional events. Mutations eliminating potential transcription factor binding sites, including the multiple-response element (MRE), AP-1, NF-IL6, and NF-kappaB binding sites, significantly reduce, but do not completely abrogate, inducibility by cAMP or prostaglandin E1, whereas alterations of four additional putative regulatory elements have no effects. In contrast, LPS-induced promoter activity is almost completely abolished by mutations in the NF-kappaB binding site, suggesting that a single regulatory element is crucial for inducibility by LPS, whereas no individual element is absolutely essential for cAMP signaling. Induction of the AP-1, NF-IL-6, and NF-kappaB elements by cAMP is correlated with the appearance of inducible factors binding to these sites, whereas factors binding to the MRE are constitutively expressed. Our results suggest that cAMP and prostaglandins act through multiple, partially redundant, regulatory elements to induce IL-6 expression in monocytic cells.