Previous studies in adult myocytes isolated from rat hearts 3-9 weeks after myocardial infarction (MI) demonstrated abnormal contractility and decreased Na⁺/Ca²⁺ exchange (NCX1) activity. In addition, a program of high intensity sprint training (HIST) instituted shortly after MI restored both contractility and NCX1 activity towards normal. The present study examined the hypotheses that reduced NCX1 activity caused abnormal contractility in myocytes isolated from sedentary (Sed) rat hearts 9-11 weeks after coronary artery ligation, and that HIST ameliorated contractile dysfunction in post-MI myocytes by increasing NCX1 activity. The approach was to upregulate NCX1 in MISed myocytes and downregulate NCX1 in MIHIST myocytes by adenovirus mediated gene transfer. Three days after infection with the control adenovirus expressing green fluorescent protein (GFP), differences in contraction amplitudes among Sham, MISed, and MIHIST myocytes were maintained. Overexpression of NCX1 in MISed myocytes did not affect sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA2) and calsequestrin levels but rescued contractile abnormalities observed in MISed myocytes. That is, at 5 mM [Ca²⁺]_o, the subnormal contraction amplitude in MISed myocytes (compared with Sham myocytes) was increased towards normal by NCX1 overexpression whereas at 0.6 mM [Ca²⁺]_o, the supernormal contraction amplitude in MISed myocytes was lowered. Conversely, NCX1 downregulation by antisense (AS) in MIHIST myocytes abolished the beneficial effects of HIST on contraction amplitudes in MI myocytes. Comparison of contraction amplitudes among control Sham, MIHIST, and MISed myocytes overexpressing NCX1 showed no significant differences. Similarly, contraction amplitudes between MISed and MIHIST myocytes in which NCX1 was downregulated were similar. We suggest that decreased NCX1 activity may play an important role in contractile abnormalities in post-MI myocytes, and that HIST ameliorated contractile dysfunction in post-MI myocytes partly by enhancing NCX1 activity.