To investigate the regulation of native cardiac Na+-Ca2+ exchange by cytoplasmic Na+ (Na+i) and Ca2+ (Ca2+i), we recorded the Na+-Ca2+ exchange current (INa-Ca) from inside-out 'macro patches' excised from intact guinea-pig ventricular cells. The half-maximal concentration (Kh) of Ca2+i required to induce an inward INa-Ca was 7 µM. The Kh of Na+i required to induce an outward INa-Ca was 21 mM, and tended to decrease at the steady state of Na+-dependent inactivation. The time constant (tau) of Na+-dependent inactivation was ~1.5 s at 100 mM Na+i and 1 µM Ca2+i. The Kh for Na+i was 14 mM. Ca2+i augmented the peak outward INa-Ca (Kh = 0.2 µM) and attenuated Na+-dependent inactivation (Kh = 2.2 µM). The outward INa-Ca was activated by 5 µM Ca2+i with a half-time to reach steady state (t½) of ~0.4 s. This activation was composed of two exponential processes. Deactivation of the current upon Ca2+i removal also consisted of two exponential processes and had a t½ of ~0.5 s. A Na+-Ca2+ exchange model, consisting of one consecutive 4Na+:1Ca2+ exchange cycle and two inactive states, well mimicked the experimental data with regard to ion dependencies and regulation kinetics. These data provide detailed information on the kinetics of the Na+i- and Ca2+i-dependent regulation of native Na+-Ca2+ exchange. They also indicate that the regulation kinetics operate faster in macro patches than in the giant membrane patch from cardiac 'blebs', or in Xenopus oocytes expressing a cloned exchanger (NCX1.1).