Ca2+ binding and concentrations of non-esterified fatty acids and phospholipids were compared in membrane fractions of rat small intestine. These fractions differed in density and were enriched for galactosyltransferase activity, a Golgi-membrane marker. Ca2+ binding was highest in the Golgi subfraction with the least density, as were the concentrations of both non-esterified fatty acids and phospholipids; galactosyltransferase activity was distributed differently. The large amount of non-esterified fatty acids was sufficient to account for a 2:1 complex of fatty acid-Ca2+. In vitamin D-deficient animals, the yield of protein in the lightest subfractions was decreased, but Ca2+ binding per mg of protein was further decreased to about 60%. In Golgi fractions from vitamin D-deficient animals, Ca2+ binding and the concentration of non-esterified fatty acids were decreased in parallel, but phospholipids were not significantly changed. There was a close correlation between Golgi Ca2+ binding and non-esterified fatty acid concentrations (r = 0.89; P less than 0.001). Non-esterified fatty acids, which are unusually prevalent in these membrane fractions, are likely to be the binding sites that account for this vitamin D-dependent Ca2+ uptake.