Hyperglycemia diminishes positive inotropic responses to agonists that activate phospholipase C (PLC) and generate inositol trisphosphate (1,4,5). The mechanisms underlying both the inotropic responses and hyperglycemia's effects on them remain undetermined, but data from isolated cardiomyocytes suggest the involvement of capacitative Ca2+ entry (CCE), the influx of Ca 2+ through plasma membrane channels activated in response to depletion of endoplasmic or sarcoplasmic reticulum Ca2+ stores. In neonatal rat cardiomyocytes, hyperglycemia decreased CCE induced by PLC-mediated agonists. The attenuation of CCE was also seen with glucosamine, and the inhibition by hyperglycemia was prevented by azaserine, thereby implicating hexosamine biosynthesis as the responsible metabolic pathway. In the current study, the importance of hexosamine metabolites to hyperglycemia's effects on inotropic responses was examined in isolated perfused rat hearts. The inhibition by hyperglycemia of phenylephrine-induced inotropy was reversed with azaserine and mimicked by glucosamine. An independent inhibitor of CCE, SKF96365, was also effective in blunting inotropy. These treatments did not inhibit inotropy induced by activation of adenylate cyclase through β-adrenergic receptors. These data thus implicate CCE in responses to PLC-mediated agonists in the intact heart and point to the hexosamine pathway's negative effect on CCE as being central to the inhibition seen with hyperglycemia.