Trimetazidine (TMZ) added in vitro to isolated cardiac mitochondria at concentrations 10-100 microM in the presence of 25-100 nM extramitochondrial Ca2+ increased Ca2+ uptake and matrix Ca2+ concentration. This effect was less evident in the presence of physiologically Na+ and Mg2+ extramitochondrial concentrations since only 100 microM TMZ was able to increase mitochondrial Ca2+ entry in the presence of 100 nM Ca2+. The drug stimulated a Ca(2+)-cooperative effect on mitochondrial Ca2+ transport, but did not modify the rate of Ca2+ egress stimulated by 10 mM NaCl. An increase in mitochondrial Ca2+ level produced by TMZ enhanced oxoglutarate dehydrogenase activity and then ATP synthesis, particularly when 50 nM extramitochondrial Ca2+ was used. These data suggest that a possible cardiac mechanism of action of TMZ at mitochondrial level could support ATP synthesis by elevating the mitochondrial Ca2+ level.