Calcium ion decreases the lipid fluidity of isolated rat hepatocyte plasma membranes by modulating the activity of membrane enzymes which alter the lipid composition. To explore the mechanism of the effect of the cation, eight fluorophores were used to assess lipid fluidity via estimations of either steady-state fluorescence polarization or excimer fluorescence intensity. The results demonstrate that the reduction in fluidity occurs in the hydrophobic interior of the bilayer and that both the dynamic and static (lipid order) components of fluidity are affected by treatment with calcium. Analysis of the membrane lipids demonstrates that calcium treatment decreases the arachidonic acid content of the polar lipid fraction and, thereby, reduces the double-bond index of the fatty acids. This change in composition, which is expected to reduce the lipid fluidity, may result from activation by calcium of the endogenous hepatocyte plasma membrane phospholipase A2.