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 (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 CO and its second messenger cGMP, 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 medium (pH 6.8) significantly increased HO-1 mRNA and protein compared with exposure to medium of physiological 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 posttranscriptionally upregulated by EA in VSMCs. This induction is time dependent and reversible. We speculate that EA, as an important tissue and cellular stressor for VSMCs, may elicit changes in gene expression patterns that contribute to the maintenance or disruption of vascular homeostasis.