Membrane fractionation studies using circular and longitudinal muscles from rabbit small intestine showed that optimal methods for preparation of microsomes and their fractionation on discontinuous sucrose density gradients were identical for the two muscles. 5′-Nucleotidase was maximally enriched in the F2 (20–28% (w/w) sucrose) fraction, NADPH: cytochrome c oxidoreductase was maximal in the F3 Fraction, and cytochrome oxidase specific activity was highest in the mitochondrial fraction. The ATP-dependent Ca uptake was highest in the F2 fraction. All membrane fractions, including F2, from the longitudinal muscle exhibited much higher ATP-dependent Ca uptake than the corresponding fractions from the circular muscle. Azide inhibited Ca uptake, in the presence of ATP, by mitochondrial fractions from the longitudinal muscle. Oxalate potentiated the ATP-dependent Ca uptake by the various subcellular fractions from the longitudinal muscle. Azide and oxalate had no effects on Ca uptake by any of the fractions derived from the circular muscle. A23187 abolished the Ca uptake in presence of ATP by F2 from either muscle. When F2 vesicles after Ca uptake in presence of ATP were diluted in hypotonic EGTA, they lost 70–80% of the stored Ca in <10 s. However, a similar dilution in isotonic EGTA led to significantly smaller loss of the stored Ca in <10 s and the remainder was lost with a half-life of 5.6 min from the circular muscle F2 vesicles and 6.2 min from the longitudinal muscle F2 vesicles. Passive Ca uptake capacities of F2 from the two muscles were similar. Thus the higher Ca uptake in presence of ATP, by longitudinal muscle F2 vesicles than by the circular muscle F2 vesicles, may be due to a higher specific activity of the Ca pump in the longitudinal muscle than in the circular muscle. The contribution due to differences in intactness and orientation of the vesicles in the two muscles is unknown at this time.