Postnatal maturation of rat heart is characterized by major changes in the mechanism of excitation-contraction (E-C) coupling. In neonate, the T tubules and sarcoplasmic reticulum (SR) are not fully developed yet. Consequently, Ca²⁺ induced Ca²⁺ release (CICR) does not play a central role in E-C coupling. In neonate, most of the Ca²⁺ that triggers contraction comes through the sarcolemma. In this work, we defined the contribution of the sarcolemmal Ca²⁺ entry and the Ca²⁺ released from the SR to the Ca²⁺ transient during the first three weeks of postnatal development. To this end, intracellular Ca²⁺ transients were measured in whole hearts from neonate rats using the pulsed local- field fluorescence technique. To estimate the contribution of each Ca²⁺ flux to the global intracellular Ca²⁺ transient different pharmacological agents were used. Ryanodine was applied to evaluate ryanodine receptor (RyR) -mediated Ca²⁺ release from the SR, nifedipine for dihydropyridine-sensitive L-type Ca²⁺ current, Ni²⁺ for the current resulting from the reverse-mode Na⁺/Ca²⁺ exchange, and mibefradil for the T-type Ca²⁺ current. Our results showed that the relative contribution of each Ca²⁺ flux changes considerably during the first three weeks of postnatal development. Early after birth (1-5 days), the sarcolemmal Ca²⁺ flux predominates, while at 3 weeks of age CICR from the SR is the most important. This transition may reflect the progressive development of the T-tube-SR units characteristic of mature myocytes. We have hence, directly defined in the whole beating heart, the developmental changes of E-C coupling previously evaluated in single/cultured cells and multicellular preparations.