Calcium ions play an important role in several cell functions, from fertilization to cell death. The cytosolic Ca²⁺ concentration is much lower than the extracellular concentration ([Ca²⁺]_o). The latter may markedly affect Ca²⁺ fluxes across the cell membrane and thus the cellular Ca²⁺ load. Thus, when working with preparations in vitro, it is important to keep [Ca²⁺]_o close to the in vivo value. In this study, we determined the calcemia in immature rats, for which values are currently unavailable, and investigated how supraphysiological [Ca²⁺]_o affects myocardial Ca²⁺ handling. Blood ionized [Ca²⁺] was similar in neonatal (2–5 days old) and adults Wistar rats (1.28 ± 0.03 and 1.31 ± 0.03 mmol/l; n=6 and 5, respectively, P>0.37), and lower than the [Ca²⁺]_o range often used in experiments with neonatal myocardial preparations. Cytosolic Ca²⁺ transients, measured with indo-1 in neonatal ventricular myocytes, were enhanced by an increase in [Ca²⁺] _o from 1.2 to 2 mM, which also increased the Ca²⁺ content in the sarcoplasmic reticulum (SR), and changed the pattern of competition between the main transporters that remove Ca²⁺ from the cytosol (SR Ca²⁺-ATPase and Na⁺/Ca²⁺ exchanger). These observations stress the importance of using physiological [Ca²⁺]_o values for reliability of results. It is expected that the present calcemia data, reported for the first time in immature rats, may contribute to the refinement of in vitro experiments with neonatal rat preparations.