Adsorption of caltrin on a cation exchanger during purification transformed it from inhibitor to enhancer of calcium uptake. Ether extracts of acidified preparations of bovine seminal plasma transformed enhancer caltrin back to inhibitory caltrin; this capacity of the ether extracts was lost after incubation with an anion exchanger, indicating that anions in the extract could be responsible for the reversal of caltrin activity. Of the anions identified in the ether extract of acidified bovine seminal plasma, phosphatidylserine converted enhancer caltrin to the inhibitory form at pH 7.4. Citrate at millimolar concentrations lowered calcium uptake of sperm in the presence of enhancer caltrin to near control levels. Cardiolipin at concentrations comparable to its natural occurrence in the seminal plasma prevented enhancer caltrin from stimulating the sperm cells to take up calcium above their usual capacity. Dipalmitoylphosphatidylglycerol, phosphatidylcholine derived from bovine brain, phosphatidylethanolamine from bovine heart, and other phospholipids with transition temperatures higher than the assay temperature had no effect on the activity of enhancer caltrin, while dimyristoylphosphatidylcholine had an effect on enhancer caltrin similar to that of citrate. Phosphatidylinositol from soybean was also capable of lowering caltrin-stimulated calcium uptake in bovine sperm to control levels. Data on enhancer caltrin fluorescence in the presence of phosphatidylserine from bovine brain suggest conformational changes in the protein due to binding of the phospholipid. In comparison, the phosphatidylcholine from bovine brain appeared not to alter enhancer caltrin.