Ca/+ transport and respiratory characteristics of two preparations of cardiac mitochondria (Palmer, J.W., Tandler, B. and Hoppel, C.L. (1977) J. Biol. Chem. 252, 8731-8739) isolated using polytron homogenization (subsarcolemmal mitochondria) and limited Nagarse exposure (intermyofibrillar mitochondria) are described. The Nagarse procedure yields mitochondria with 50% higher rates of oxidative phosphorylation than the polytron-prepared mitochondria in both rat and dog. Rat hear intermyofibrillar mitochondria contain 50% more cytochrome aa3 than the polytron preparation, whereas in the dog, cytochrome aa3 content is not significantly different. Cytochrome oxidase activities and cytochrome c, c1 and b contents were comparable in both populations of rat and dog heart mitochondria. The V of succinate-supported Ca2+ accumulation for Nagarse-prepared mitochondria from rat heart was 1.8-fold higher than the polytron-prepared mitochondria. In dog heart, the Nagarse preparation showed a 3.0-fold higher V for Ca2+ uptake compared to the polytron preparation. A lower apparent affinity for Ca2+ was demonstrated in the intermyofibrillar mitochondria for both species (Km is 2-2.5-fold higher). The Hill coefficient was 1 both mitochondrial types. Subsarcolemmal mitochondria from both species were treated with Nagarse to determine the role of this treatment on the observed differences. Nagarse did not alter any kinetic parameter of Ca2+ uptake. The properties of these mitochondria with reference to their presumed intracellular location may pertain to the role of mitochondria as an intracellular Ca2+ buffering mechanism in contractile tissue.