The effect of exercise training on myocardial Na(+)/H(+) exchanger-1 (NHE1) protein expression was examined. Adult female Sprague-Dawley rats were randomly divided into sedentary (S; n = 8) and exercised (E; n = 9) groups. Twenty-four hours after the last exercise bout, hearts were weighed and connected to an isolated perfused working heart apparatus for evaluation of cardiac functional performance. Heart weight and heart weight/body weight from E rats was significantly increased by 7.1 and 7.2 % (P < 0.05), respectively, compared with S hearts. The E hearts displayed 15 % greater cardiac output and 35 % external cardiac work compared with the S group at both low and high workloads (P < 0.05 for both parameters). Left ventricular tissue from the same hearts was homogenized and NHE1 and Na(+)/Ca(2+) exchanger (NCX) content determined by Western blotting. E hearts had a 38 % (P < 0.001) reduction in NHE1 content related to S hearts, and there was no difference in NCX content between groups. Cytochrome c oxidase activity in plantaris increased by 100 % (P < 0.05) and was assessed as a marker of mitochondria content and to verify training status. Our data indicate that exercise training at an intensity that results in cardiac hypertrophy and improved performance is accompanied by decreased NHE1 content in heart.