A novel liposomal method permits studies of Ca movements across the bilayers of multilamellar vesicles (MLV) which had entrapped the Ca-dependent, fluorescent indicator dye Fura 2. Ionomycin-mediated Ca translocation across MLV of phosphatidylcholine (PC)/dicetyl phosphate (DCP), 9:1, obeyed simple first-order kinetics since log-log plots of initial rates versus ionomycin or Ca concentration yielded slopes of approximately 1. Since Ca is translocated in a Ca-dependent fashion in the course of stimulus-response coupling of cells which form diacylglycerol (DAG) and phosphatidate (PA) from polyphosphoinostidies, we compared effects of PA with those of DAG. PA and DAG were preincorporated in PC/DCP vesicles, in which trace amounts of ionomycin provided transmembrane potential (due to Ca2+/H+ exchange). Significant increases in Ca movements were observed in the presence of egg lecithin PA, dioleoyl-PA, and dipalmitoyl-PA when compared with DCP- or DAG-containing MLV., DAGs such as 1-oleoyl-2-acetoylglycerol or 1,2-dioleoylglycerol in liposomes decreased rates of Ca translocation. Ca influx into PA-containing MLV was dependent on the mole percent of the PA in bilayers; the complex kinetics of Ca influx were compatible with the formation of nonbilayer states. Incorporation of cholesterol into the liposomes inhibited initial rates of Ca uptake by MLV presumably by condensing the bilayers. Ca influx increased with increasing pH of the external medium from 6.9 to 7.9 in liposomes with an internal pH of 7.4. The results not only indicate that transmembrane pH gradients and the extent of ionization of the ionophore affect rates of Ca translocation across lipid bilayers but also demonstrate that in model systems PA but not DAG promotes Ca translocation, the rate of which is altered by lipid composition of the bilayer and the pH of the suspension.