Reverse-mode activity of the Na+/Ca2+exchanger (NCX) has been previously shown to play a prominent role in excitation-contraction coupling in the neonatal rabbit heart, where we have proposed that a restricted subsarcolemmal domain allows a Na+current to cause an elevation in the Na+concentration sufficiently large to bring Ca2+into the myocyte through reverse-mode NCX. In the present study, we tested the hypothesis that there is an overlapping expression and distribution of voltage-gated Na+(Nav) channel isoforms and the NCX in the neonatal heart. For this purpose, Western blot analysis, immunocytochemistry, confocal microscopy, and image analyses were used. Here, we report the robust expression of skeletal Nav1.4 and cardiac Nav1.5 in neonatal myocytes. Both isoforms colocalized with the NCX, and Nav1.5-NCX colocalization was not statistically different from Nav1.4-NCX colocalization in the neonatal group. Western blot analysis also showed that Nav1.4 expression decreased by sixfold in the adult ( P < 0.01) and Nav1.1 expression decreased by ninefold ( P < 0.01), whereas Nav1.5 expression did not change. Although Nav1.4 underwent large changes in expression levels, the Nav1.4-NCX colocalization relationship did not change with age. In contrast, Nav1.5-NCX colocalization decreased ∼50% with development. Distance analysis indicated that the decrease in Nav1.5-NCX colocalization occurs due to a statistically significant increase in separation distances between Nav1.5 and NCX objects. Taken together, the robust expression of both Nav1.4 and Nav1.5 isoforms and their colocalization with the NCX in the neonatal heart provides structural support for Na+current-induced Ca2+entry through reverse-mode NCX. In contrast, this mechanism is likely less efficient in the adult heart because the expression of Nav1.4 and NCX is lower and the separation distance between Nav1.5 and NCX is larger.