Exposure of rats to 10% O(2) for 4 days caused pulmonary hypertension and induced expression of both inducible nitric oxide synthase (iNOS) and CCAAT box enhancer binding protein-beta (C/EBP-beta) in rat lung. Electrophoretic mobility shift assays (EMSAs) showed that exposure to 1% O(2) increased the C/EBP-beta binding in rat pulmonary microvascular smooth muscle cells (rPSMs). To test the hypothesis that C/EBP-beta participates in hypoxia-induced iNOS expression in rPSMs, a C/EBP motif at -910 bp of rat iNOS promoter was mutated. rPSMs transfected with the rat iNOS promoter and exposed to 1% O(2) for 24 hours had significantly increased wild-type iNOS promoter activity. The hypoxia-induced promoter activity was abolished by the C/EBP motif mutation. Thus, C/EBP-beta mediates, at least in part, hypoxia-induced iNOS expression in rPSMs.