Adult rat cardiac myocytes typically display a phenotypic response to cytokines manifested by low or no increases in nitric oxide (NO) production via inducible NO synthase (iNOS) that distinguishes it from other cell types. To better characterize this response, we examined expression of tetrahydrobiopterin (BH₄)-synthesizing and arginine-utilizing genes in cytokine-stimulated, adult cardiac myocytes. Intracellular BH₄ and 7,8-dihydrobiopterin (BH₂) and NO production were quantified. Cytokines induced GTP cyclohydrolase and its feedback regulatory protein but with deficient levels of BH₄ synthesis. Despite induction of iNOS protein, cytokine-stimulated adult cardiac myocytes produced little or no increase in NO vs unstimulated cells. Western analysis under non-reducing conditions revealed the presence of iNOS monomers. Supplementation with sepiapterin (a precursor of BH₄) increased BH₄ as well as BH₂ but this did not enhance NO levels nor eliminate iNOS monomers. Similar findings were confirmed in vivo following treatment of rat cardiac allograft recipients with sepiapterin. It was found that expression of dihydrofolate reductase required for full activity of the salvage pathway was not detected in adult cardiac myocytes. Thus adult cardiac myocytes have a limited capacity to synthesize BH₄ following cytokine stimulation. The mechanisms involve post-translational factors impairing de novo and salvage pathways. These conditions are unable to support active iNOS protein dimers necessary for NO production. These findings raise significant new questions about the prevailing understanding of how cytokines via iNOS causes cardiac dysfunction and injury in vivo during cardiac inflammatory disease states since cardiac myocytes are not a major source of high NO production.