A number of long-chain fatty acids and fatty acid derivatives accumulate in the cytosol of ischemic myocardium. Although the functional significance of this accumulation in vivo remains unclear, these amphiphilic compounds may alter the functional properties of a variety of biological membranes in vitro. In this study, we investigated the effect of palmitic acid and palmityl carnitine on calcium sequestration by sarcoplasmic reticulum vesicles in the absence of calcium-precipitating anions. Palmitic acid, at micromolar concentrations, enhanced calcium sequestration in a concentration-dependent manner when present from the onset of the reaction or when added to calcium-filled vesicles. Under identical conditions, similar concentrations of palmityl carnitine inhibited calcium sequestration when present at the onset of the reaction and caused a rapid release of accumulated calcium when added to calcium-filled vesicles. Low concentrations of palmitic acid decreased the sensitivity of the sarcoplasmic reticulum to the inhibitory effects of palmityl carnitine. These results suggest that calcium pump function in the sarcoplasmic reticulum can be altered by the presence of amphiphilic compounds and that this alteration is dependent on both the structure and number of amphiphiles present.