Sex-related differences in cardiac function have been well documented. The extent to which sex hormones are responsible for these differences is unclear. The current study was designed to determine whether castration and androgen replacement resulted in changes in functional expression of genes encoding the L-type calcium channel and Na/Ca exchanger in isolated rat ventricular myocytes. Sixteen weeks of castration produced a 50% decline in dihydropyridine receptor expression levels and a 16% (P < 0.05) increase in time to peak shortening. Furthermore, cardiac myocytes isolated from castrated animals also displayed an 18% (P < 0.001) increase in time to relengthening and an 80% decrease in Na/Ca exchanger gene expression when compared with intact controls. Testosterone treatment of castrated animals completely reversed these effects. These results provide the first evidence that androgens regulate functional expression of the L-type calcium channel and the Na/Ca exchanger in isolated rat ventricular myocytes and thus may play a role in modulating cardiac performance in males and thereby contribute to the observed gender differences in cardiac function.