Cardiac sarcoplasmic reticulum (CSR), isolated from dog hearts, was shown to be asymmetric in the distribution of phospholipids across the CSR bilayer. Phosphatidylethanolamine was mostly resident in the outer leaflet, phosphatidylcholine was equally distributed across both monolayers and phosphatidylserine was found primarily in the inner monolayer. This distribution of headgroups is similar to that found in fast skeletal muscle sarcoplasmic reticulum (SSR); however, the asymmetry in CSR is not as striking as that in SSR. Phospholipids retained by the CSR calcium pump protein (CaATPase) after detergent "stripping" were similar to those intimate to the SSR CaATPase, although the percentages of unsaturated phospholipids and plasmalogenic phospholipids are not as great as in the skeletal system. Lipids associated with the CSR CaATPase following DFDNB cross-linking showed a preference for retention of the aminophospholipids, again similar to the SSR CaATPase. Because the nonrandom distribution of membrane lipids modifies SSR function, it is likely these membrane lipids impact in situ the function of the CSR.