Previous studies have shown that Ca2+ channel antagonists in all chemical classes can inhibit Na(+)-induced CA2+ release from mitochondria. The effects of R023-6152, a new thiazepinone Ca2+ channel antagonist, on isolated rabbit heart mitochondrial Ca2+ transport and respiratory activity were compared with those of diltiazem. Heart mitochondria were also isolated and assayed from dogs treated in vivo with either R023-6152 or diltiazem. The results indicate that R023-6152 produces half-maximal inhibition of Na(+)-induced Ca2+ release from isolated mitochondria at relatively the same concentrations (10-30 microM) as diltiazem but also produces inhibition of mitochondrial Ca2+ uptake and state 3 respiration at concentrations (25-100 microM), at which diltiazem has no effect. The greater lipophilicity of R023-6152 in gaining access to and inhibiting the phosphate transporter in the mitochondrial membrane as compared with that of diltiazem may explain these results. Heart mitochondria isolated from dogs treated with diltiazem and R023-6152 exhibited lower rates of state 3 respiration as compared with controls. We suggest that this may result from a reduction in transsarcolemmal Ca2+ flux causing a down-regulation in mitochondrial dehydrogenase activity and not from any direct intracellular effects of the two drugs.