Extracellular acidosis (EA) has profound effects on vascular homeostasis, including vascular-bed specific alterations in vascular tone. Regulation of gene expression by EA has been observed in a variety of cells including vascular endothelial cells. Whether EA regulates gene expression in vascular smooth muscle cells (VSMCs) is not known. Heme oxygenase-1 (HO-1) is expressed in vascular cells and its expression is regulated by cellular stressors such as heat, radiation and hypoxia. Increased HO-1 expression in VSMCs leads to increased production of carbon monoxide (CO) and its second messenger cyclic GMP which are important regulators of vascular tone and paracrine interactions in the vasculature. We examined whether EA regulates the expression of HO-1 in VSMCs. Exposure of VSMCs to acidic media (pH 6.8) significantly increased HO-1 mRNA and protein compared to exposure to media of physiologic pH (pH 7.4). The acidic-induction of HO-1 expression was time-dependent and involved both transcriptional activation of the HO-1 gene and enhanced stability of HO-1 mRNA. Nitric oxide did not appear to mediate this response. We conclude that HO-1 is transcriptionally and posttranscrtiptionally up-regulated by extracellular acidosis in VSMCs. This induction is time-dependent and reversible. We speculate that extracellular acidosis, as an important tissue and cellular stressor for vascular smooth muscle cells may elicit changes in gene expression patterns that contribute to the maintenance or disruption of vascular homeostasis.