The relative importance of the Na⁺/Ca²⁺exchanger in the initial and terminal phases of relaxation and the decline in the [Ca²⁺]_itransient was investigated in adult rabbit ventricular myocytes loaded with the Ca²⁺ indicator fluo 3. For electrically stimulated contractions, the peak intracellular Ca²⁺ concentration ([Ca²⁺]_i) was 700 ± 87 nM and end-diastolic [Ca²⁺]_iwas 239 ± 30 nM (0.25 Hz, 37°C, 1.08 mM extracellular Ca²⁺ concentration;n = 14). Abrupt inhibition of Na⁺/Ca²⁺exchange was produced by removal of extracellular Na⁺ (KCl substitution) and Ca²⁺ [2 mM Ca²⁺-free ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid] by means of a rapid switcher device (SW). Abrupt exposure to high K⁺ induced an action potential, although sufficient Ca²⁺ remained adjacent to the sarcolemma to induce a contraction (SW beat) and [Ca²⁺]_itransient that were identical in amplitude to those induced by electrical stimulation (ES beat). The initial relaxation and decline in the [Ca²⁺]_itransient was not significantly prolonged by abrupt elimination of the Na⁺/Ca²⁺exchanger, but the rate and extent of the terminal phase of the decline in the [Ca²⁺]_itransient were significantly reduced. The first derivative of [Ca²⁺]_iwith respect to time versus [Ca²⁺]_iduring the decline of the [Ca²⁺]_itransient attributable to sarcoplasmic reticulum (SR) function was estimated from the average SW transients, and that attributable to Na⁺/Ca²⁺exchange was estimated from the difference between SW and ES transients. By this analysis, the Na⁺/Ca²⁺exchanger produces 13% of the first half of the decline in [Ca²⁺]_iand 45% of the second half of the decline. We conclude that abrupt inhibition of forward Na⁺/Ca²⁺exchange does not significantly affect the amplitude or the initial rate of decline of the [Ca²⁺]_itransient and relaxation. However, its contribution to the reduction of [Ca²⁺]_ibecomes apparent late during the [Ca²⁺]_itransient, when cytosolic [Ca²⁺]_ihas been reduced.