Subcellular membrane fractions were isolated from the circular muscle of the corpus of canine stomach by differential and isopycnic sucrose density gradient centrifugation. Differential centrifugation gave a mitochondrial fraction enriched (fourfold) in cytochrome c oxidase and a microsomal fraction enriched (fourfold) in 5′-nucleotidase and NADPH–cytochrome c reductase over postnuclear supernatant. On the basis of a study using continuous gradient, a discontinuous sucrose density gradient was prepared to yield F1 to F5 fractions. The F3 fraction at the interface of 18–32% (w/w) sucrose was maximally enriched (13-fold) in 5′-nucleotidase. The fraction contained very low levels of cytochrome c oxidase but did contain NADPH–cytochrome c reductase (eightfold enrichment). The F4 fraction, at the interface of 32–40% (w/w) sucrose, was maximally enriched in NADPH–cytochrome c reductase (12-fold) and cytochrome c oxidase (6-fold). The distribution of the azide-insensitive, ATP-dependent Ca2+ uptake correlated very well with that of 5′-nucleotidase but less well with NADPH–cytochrome c reductase and not at all with cytochrome c oxidase. Sodium azide and ruthenium red inhibited the ATP-dependent Ca2+ uptake by the mitochondrial fraction and postnuclear supernatant, but not by the F3 fraction. ATP-dependent Ca2+ uptake by the F3 fraction was inhibited by calcium ionophores A23187 and ionomycin, but not by the sodium ionophore, monensin. These results are consistent with the hypothesis that the plasma membrane plays a major role in regulating intracellular Ca2+ concentration in canine corpus circular muscle.