In the present paper we describe a method to estimate mitochondrial Ca2+ uptake during the declining phase of Ca2+ transients (cell relaxation) in intact isolated myocardial cells. This method is based on inhibition of sarcoplasmic reticulum (SR) Ca2+ accumulation by caffeine, blockade of Ca2+ transport via sarcolemmal Ca(2+)-ATPase by treatment with carboxyeosin and inhibition of sarcolemmal Na+/Ca2+ exchange by removal of extracellular Na+ and Ca2+.Ca2+ transients were evoked in rabbit ventricular myocytes by quick and sustained caffeine application (10 mM) after a 5-min period of electrical stimulation to load the SR with Ca2+. Mitochondrial Ca2+ transport was estimated using a model described by Sipido and Wier (Journal of Physiology (1991), 435: 605-630), which was originally proposed to describe Ca2+ fluxes during excitation-contraction coupling in cardiac cells. Our results indicate that, in intact rabbit myocytes, the Ca2+ flux due to net mitochondrial Ca2+ uptake may attain a value close to 1 microM/sec.291699-707