Increased synthesis of stress proteins may enhance myocardial viability during periods of low oxygen delivery. Our purpose was to determine if the oxidative stress protein heme oxygenase-1 [heat stress protein 32 (HSP 32)] was induced in hypoxic cardiomyocytes and whether this induction might be mediated by a redox-sensitive mechanism. Primary rat neonatal cardiomyocytes, cultured to express a tissuelike phenotype, responded to 12 h of hypoxia (<0.5% ambient oxygen) with an approximately fivefold (range 3- to 7.5-fold;P < 0.05) increase in HSP 32 mRNA and a threefold (P < 0.05) increase in HSP 32 protein content. Exposure to 80 μM H₂O₂for 3 h increased HSP 32 mRNA content to a similar extent. Expression of heme oxygenase-2 mRNA was unaffected by H₂O₂or hypoxic treatments. Inclusion of 20 mMN-acetyl-l-cysteine in the medium during hypoxia reduced the increase in HSP 32 mRNA and protein expression by 25–50% compared with hypoxia alone. The data suggest that induction of HSP 32 protein may lead to an improved antioxidant defense in cardiomyocytes during hypoxia and that a redox-sensitive pathway mediates at least a portion of the hypoxic induction of the HSP 32gene.