Nitric oxide (NO) has an important role in modulating the pulmonary vascular tone. NO acts, in part, by stimulating soluble guanylate cyclase (sGC) to synthesize the intracellular second messenger cyclic GMP. In vascular smooth muscle cells, sGC is a heterodimer composed of alpha1 and beta1 subunits. The objective of this study was to test whether oxygen concentration regulates sGC expression in cultured rat pulmonary artery smooth muscle cells (rPaSMC). rPaSMC were exposed to 0, 3, and 20% oxygen for 1-48 h, and sGC subunit mRNA levels were measured. Compared with rPaSMC exposed to 20% oxygen, sGC alpha1 and beta1 subunit mRNA levels were markedly decreased in rPaSMC exposed to 0% and 3% oxygen. The decrease in sGC subunit mRNA levels in hypoxic rPaSMC was detected as early as 6 h of exposure. Compared with rPaSMC exposed to 20% oxygen, exposure of rPaSMC to 3% oxygen progressively decreased sGC subunit protein levels at 24 and 48 h. There was also a 30% and 50% decrease in sGC enzyme activity in cells exposed to hypoxia for 24 and 48 h (P < 0.05 and P < 0.001, respectively, as compared with cells maintained in normoxia). These results demonstrate that hypoxia decreases sGC expression in cultured pulmonary artery smooth muscle cells and suggest that, in hypoxic vascular smooth muscle, decreased cyclic GMP synthesis may limit the vasodilator response to NO.