Mechanisms underlying the negative inotropic response to α-adrenoceptor stimulation in adult mouse ventricular myocardium were studied. In isolated ventricular tissue, phenylephrine (PE), in the presence of propranolol, decreased contractile force by ∼40% of basal value. The negative inotropic response was similarly observed under low extracellular Ca²⁺ concentration ([Ca²⁺]_o) conditions but was significantly smaller under high-[Ca²⁺]_o conditions and was not observed under low-[Na⁺]_o conditions. The negative inotropic response was not affected by nicardipine, ryanodine, ouabain, or dimethylamiloride (DMA), inhibitors of L-type Ca²⁺ channel, Ca²⁺ release channel, Na⁺-K⁺ pump, or Na⁺/H⁺exchanger, respectively. KB-R7943, an inhibitor of Na⁺/Ca²⁺ exchanger, suppressed the negative inotropic response mediated by PE. PE reduced the magnitude of postrest contractions. PE caused a decrease in duration of the late plateau phase of action potential and a slight increase in resting membrane potential; time courses of these effects were similar to that of the negative inotropic effect. In whole cell voltage-clamped myocytes, PE increased the L-type Ca²⁺ and Na⁺/Ca²⁺ exchanger currents but had no effect on the inwardly rectifying K⁺, transient outward K⁺, or Na⁺-K⁺-pump currents. These results suggest that the sustained negative inotropic response to α-adrenoceptor stimulation of adult mouse ventricular myocardium is mediated by enhancement of Ca²⁺ efflux through the Na⁺/Ca²⁺ exchanger.