Surfactant protein B (SP-B) is an essential constituent of pulmonary surfactant. In a number of inflammatory diseases of the lung, elevated nitric oxide (NO) levels are associated with decreased SP-B levels, suggesting that reduced SP-B levels contribute to lung injury. In this study, we investigated the effects of NO on SP-B gene expression in H441 and MLE-12 cells, cell lines with characteristics of bronchiolar (Clara) and alveolar type II epithelial cells, respectively. Results show that NO donors decreased SP-B mRNA levels in a concentration- and time-dependent manner in H441 and MLE-12 cells. The NO donors also antagonized dexamethasone induction of SP-B mRNA in H441 cells. NO donor inhibition of SP-B mRNA was blocked by the transcriptional inhibitor 5,6-dichloro-1-beta-D-ribofuranozyl-benzimidazole. NO donors decreased luciferase expression from a reporter plasmid containing -911/+41 bp of human SP-B 5'-flanking DNA in H441 and MLE-12 cells, indicating inhibitory effects on SP-B promoter activity. NO inhibition of SP-B mRNA levels was not blocked by LY-83583 and KT-5823, inhibitors of soluble guanylate cyclase and protein kinase G, respectively. Furthermore, cell-permeable cGMP analog 8-bromo-cGMP had no effect on SP-B mRNA levels. These data indicate that elevated NO levels negatively regulate SP-B gene expression by inhibiting gene transcription and that NO inhibits SP-B gene expression independently of cGMP levels. These data imply that reduced SP-B expression due to elevated NO levels can contribute to lung injury.