Homozygous overexpression of the cardiac Na+-Ca2+ exchanger causes cardiac hypertrophy and increases susceptibility to heart failure in response to stress. We studied the functional effects of homozygous overexpression of the exchanger at the cellular level in isolated mouse ventricular myocytes. Compared with patch- clamped myocytes from wild-type animals, non-failing myocytes from homozygous transgenic mice exhibited increased cell capacitance (from 208 ± 16 pF to 260 ± 15 pF, P < 0.05). Intracellular Ca2+ oscillations were readily elicited in homozygous transgenic animals during depolarizations to +80 mV, consistent with rapid Ca2+ overload caused by reverse Na+-Ca2+ exchange. After normalization to cell capacitance, transgenic myocytes had significant increases in Na+-Ca2+ exchange activity (318%) and peak L-type Ca2+ current (8.2 ± 0.7 pA/pF at 0 mV test potential) compared to wild-type (5.8 ± 0.9 pA/pF at 0 mV, P < 0.02). The peak Ca2+ current amplitude and its rate of inactivation could be modulated by rapid reversible block of the exchanger. Thus, we describe an unexpected direct influence of Na+-Ca2+ exchange activity on the L-type Ca2+ channel. Despite intact sarcoplasmic reticular Ca2+ content and larger peak L- type Ca2+ currents, however, homozygous transgenic animals exhibited smaller Ca2+ transients ([Ca2+] i = 466 ± 48 nM in transgenics vs. 892 ± 104 nM in wild-type, P < 0.0005) and substantially reduced gain of excitation-contraction coupling. These alterations in excitation-contraction coupling may underlie the tendency for these animals to develop heart failure following hemodynamic stress.