The purpose of this study is to elucidate the mechanism of action and site of action of calcitonin gene-related peptide (CGRP) and its effects on calcium concentrations in two types of cardiomyocytes, neonatal and adult, by employing real-time fluorescence imaging. CGRP caused an increase in intramyocytic calcium with adult cells, but a decrease with neonates. Treatment of adult myocytes with ouabain and ryanodine yielded results suggesting that CGRP action is not at the ryanodine receptor (RyR) and does not involve Na + +K + ATPase. Furthermore, in neonatal cardiomyocytes CGRP caused a reduction in intramyocytic calcium levels, and challenges with ryanodine and ouabain gave results supporting the hypothesis that CGRP acts at the sarcolemmal L-type calcium channel. Employing real-time fluorescence measurements in cultured, dedifferentiated adult cardiomyocytes, which are known to express a fetal phenotype and exhibit neonatal-like calcium transients, our acquisitions demonstrated a major reduction in intracellular calcium levels. Finally, our collaborative studies in human myocardium using fluorescence deconvolution microscopy revealed that CGRP localization was found in a pattern similar to that of the sarcolemmal L-type calcium channel.