High levels of oxidative stress promote cardiac myocyte death, though lower levels are potentially cytoprotective/anabolic. We examined the changes in gene expression in rat neonatal cardiac myocytes exposed to apoptotic (0.2 mM) or nontoxic (0.04 mM) concentrations of H₂O₂ (2, 4, or 24 h) using Affymetrix microarrays. Using U34B arrays, we identified a ubiquitously expressed, novel H₂O₂-responsive gene [putative peroxide-inducible transcript 1 (Perit1)], which generates two alternatively spliced transcripts. Using 230 2.0 arrays, H₂O₂ (0.04 mM) promoted significant changes in expression of only 32 genes, all of which were seen with 0.2 mM H₂O₂. We failed to detect any increase in the rate of protein synthesis in cardiac myocytes exposed to <0.1 mM H₂O₂, further suggesting that global, low concentrations of H₂O₂ are not anabolic in this system. H₂O₂ (0.2 mM) promoted significant (P < 0.05, >1.75-fold) changes in expression of 649 mRNAs and 187 RNAs corresponding to no established gene. Of the mRNAs, 114 encoded transcriptional regulators including Krüppel-like factors (Klfs). Quantitative PCR independently verified the changes in Klf expression. Thus, H₂O₂-induced cardiac myocyte apoptosis is associated with dynamic changes in gene expression. The expression of these genes and their protein products potentially influences the progression of the apoptotic response.