Key points• L-type Ca 2+ channels are inactivated by an increase in intracellular [Ca 2+ ], known as Ca 2+ -dependent inactivation (CDI), and are inhibited by Ca 2+ released from the sarcoplasmic reticulum (SR), known as release-dependent inhibition (RDI).• RDI was greatly enhanced by the removal of Na . CDI is also induced by Ca 2+ released from the sarcoplasmic reticulum (SR), known as release-dependent inhibition (RDI). As both CDI and RDI occur in the junctional subsarcolemmal nanospace (JSS), we investigated which factors are involved within the JSS using isolated cardiac myocytes from the main pulmonary vein of the rabbit. Using the whole-cell patch clamp technique, RDI was readily observed with the application of a pre-pulse followed by a test pulse, during which the ICaLs exhibited a decrease in peak current amplitude and a slower inactivation. A fast acting Ca 2+ chelator, 1,2-bis(o-aminophenoxy)ethane-N ,N ,N ,N -tetraacetic acid (BAPTA), abolished this effect. As the time interval between the pre-pulse and test pulse increased, the ICaLs exhibited greater recovery and the RDI was relieved. Inhibition of the ryanodine receptor (RyR) to downregulate CDI and attenuates SR Ca 2+ refilling. The model may be useful to elucidate complex mechanisms involved in excitation-contraction coupling in myocytes.