Corticotropin relasing hormone (CRH) has been recently found to modulate the inflammatory response. Nitric oxide (NO) is involved in a number of pathological processes, including inflammation and genesis of tumors. The polyoma-transformed murine endothelioma cell line H5V produces NO to a larger extent than normal endothelia. We studied the effects of CRH on NO production from cultured H5V cells. Cells were incubated with graded concentrations of either CRH (100 pM-1 μM), or calcium ionophore A23187 (1-10 μM), as a stimulus for intracellular NO synthase. In addition, we investigated about the role of cyclic AMP in NO production from H5V cells, using the adenylate cyclase activator forskolin and the phosphodiesterase inhibitors theophylline (10 μM) or isobutylmethylxanthine (IBMX; 100 μM), either alone or in combination. Nitrites were measured in the medium using the Griess reaction (Green et al, Anal. Biochem. 1982). Incubation with CRH resulted in a significant decrease in production of nitrites from endothelioma cells in vitro. Similarly, either forskolin, teophyllin, or IBMX inhibited production of nitrites. Combination of CRH (100 nM) and either IBMX (100 μM) or FOR (10 μM) resulted in an additive inhibitory effect. In summary, production of nitric oxide in endothelioma cells seems partially dependent upon adenylate cyclase and intracellular calcium.