Nitric oxide (NO) production is increased in the human colonic mucosa in intestinal inflammation. We examined the effect of corticosteroids and the role of mononuclear cells in this production. Colonic biopsies from patients with ulcerative colitis and normal controls were cultured with either budesonide or prednisolone in the presence of proinflammatory cytokines. Human mixed mononuclear cells (MMCs) were co-cultured with HT-29 cells stimulated with IFN- and LPS in the presence or not of corticosteroids. Nitrite production was measured in supernatants by a modification of the Griess reaction and iNOS mRNA expression was studied in colonic tissue by RT-PCR. Both steroids significantly suppressed the nitrite production and iNOS mRNA expression in inflamed colonic biopsies from ulcerative colitis patients and in cytokine stimulated normal colonic biopsies, but not in cytokine stimulated HT-29 cells. Nitrite production by HT-29 cells was significantly increased (p<0.01) in co-cultures with MMCs stimulated with IFN- and LPS. The presence of either prednisolone or budesonide significantly (p<0.01) suppressed nitrite production from co-cultures of HT- 29 cells and MMCs, but not from cultures of HT-29 cells stimulated with conditioned media from activated MMCs. Interestingly, stimulation of HT-29 with conditioned media from MMCs pre-treated with steroids prior to stimulation with LPS and IFN- induced a significantly (p<0.01) lower nitrite production. These results suggest that the inhibitory effect of corticosteroids on the NO production in the intestinal inflammation might be via the inhibition of MMCs produced mediators responsible for NO production by colonic epithelial cells.