The intestinal mucosa is an active participant in the inflammatory and metabolic response to sepsis, endotoxemia, and other critical illness. The genes for various cytokines, e.g., interleukin 6 (IL-6), are regulated by multiple transcription factors, including nuclear factor-kappa B (NF-kappaB) and activator protein-1 (AP-1). In recent studies, treatment with IL-1beta of cultured Caco-2 cells, a human intestinal epithelial cell line, resulted in increased NF-kappaB DNA binding. The effect of IL-1beta on AP-1 activity in the enterocyte and the potential role of AP-1 in enterocyte IL-6 production are not known. We treated Caco-2 cells with IL-1beta and determined AP-1 activity by electrophoretic mobility shift assay (EMSA) and IL-6 production by enzyme-linked immunosorbent assay (ELISA). Treatment of Caco-2 cells with IL-1beta resulted in a dose- and time-dependent increase in AP-1 DNA binding. Supershift analysis suggests that activated AP-1 contained c-Jun, JunD, c-Fos, FosB, and Fra1 subunits. When Caco-2 cells were transiently transfected with an AP-1 luciferase reporter plasmid, stimulation with IL-1beta resulted in increased luciferase activity, suggesting that AP-1 DNA binding increased gene activation. Additional luciferase assays were performed with a plasmid containing a wild-type or AP-1-mutated IL-6 promoter. Stimulation of these cells with IL-1beta gave rise to results supporting the role of AP-1 in the regulation of IL-6 production. Geldanamycin, which has been shown in studies to inhibit AP-1 activation, blocked IL-1beta-induced AP-1 luciferase gene activation and IL-6 production. These results suggest that the AP-1 family of transcription factors is activated by IL-1beta in human enterocytes and that AP-1 may at least in part regulate IL-6 production in these cells.