Ca(2+) influx via sarcolemmal voltage-dependent Ca(2+) channels (L-type Ca(2+) channels) is the fundamental step in excitation-contraction (E-C) coupling in cardiac myocytes. Physiological and pharmacological studies reveal species-specific differences in E-C coupling resulting from a difference in the contribution of Ca(2+) influx and intracellular Ca(2+) release to activation of contraction. We investigated the distribution of L-type Ca(2+) channels in isolated cardiac myocytes from rabbit and rat ventricle by correlative immunoconfocal and immunogold electron microscopy. Immunofluorescence labeling revealed discrete spots in the surface plasma membrane and transverse (T) tubules in rabbit myocytes. In rat myocytes, labeling appeared more intense in T tubules than in the surface sarcolemma. Immunogold electron microscopy extended these findings, showing that the number of gold particles in the surface plasma membrane was significantly higher in rabbit than rat myocytes. In rabbit myocyte plasma membrane, the gold particles were distributed as clusters in both regions that were associated with junctional sarcoplasmic reticulum and those that were not. The findings are consistent with the idea that influx of Ca(2+) via surface sarcolemmal Ca(2+) channels contributes to intracellular Ca(2+) to a greater degree in rabbit than in rat myocytes.