The present investigations were performed to evaluate whether calcium modulates the physical state and lipid composition of rat enterocyte plasma membranes in vivo. Ca2+ [CaCl2 (50 mM) in NaCl] or NaCl (vehicle control) was administered to rats by intraluminal (gavage) or intraperitoneal routes. Sixty minutes later, brush-border (BBM) and basolateral membranes (BLM) were prepared from the proximal small intestine. By use of the fluorophores, DL-2-(9-anthroyl)-stearic acid (2-AS), DL-12-(9-anthroyl)-stearic acid (12-AS), and 1,6-diphenyl-1,3,5-hexatriene (DPH), steady-state fluorescence polarization studies demonstrated that intraluminal calcium decreased the fluidity of BBM but not BLM compared with their respective vehicle controls. These alterations in fluidity could, at least in part, be attributed to the concomitant increase in sphingomyelin content and the sphingomyelin/lecithin ratio (mol/mol) observed in the BBM prepared from calcium-gavaged rats. To evaluate the mechanism for these lipid alterations, enzyme activities involved in sphingomyelin synthesis and degradation were measured and revealed an increase in sphingomyelin synthase and a decrease in sphingomyelinase in BBM prepared from calcium-gavaged rats. In contrast, intraperitoneal administration of calcium failed to influence membrane fluidity, lipid composition, or these enzymatic activities in either BBM or BLM.