Liposomes which have entrapped the metallochromic dye, arsenazo III, constitute a sensitive assay system for ionophoresis of divalent cations. By this means we have compared known calcium ionophores (A23187, ionomycin) with membrane phospholipids, fatty acids, prostanoids, and retinoids. Added at micromolar concentrations to preformed multilamellar liposomes (phosphatidylcholine 7:dicetyl phosphate 2: cholesterol 1) both A23187 and ionomycin, as well as phosphatidic acid and products derived from linoleic acid, linolenic acid, and two eicosatrienoic acids provoked Ca influx (e.g. phosphatidic acid: 0.13 mol of Ca2+/mol of membrane lipid/5 min). A variety of other phospholipids (e.g. phosphatidylinositol), fatty acids (e.g. arachidonic acid), prostanoids (e.g. PGE1) retinoids (e.g. retinoic acid), and glyceryl ether phosphorylcholines ("platelet-activating factors") were without effect. Phosphatidic acid and oxidized fatty acids translocated divalent cations selectively, demonstrating the same rank order as A23187 or ionomycin: Mn greater than Ca greater than Sr much greater than Mg. Membrane lysis did not contribute to the perceived translocation; the liposomes remained impermeable to EDTA, EGTA, arsenazo III, or Mg. Liposomes with phosphatidic acid or oxidized trienoic acids preincorporated at 1-5 mole % of total lipids also permitted translocation of Ca but not Mg. Reduction of ionophoretic fatty acids or ionomycin with stannous chloride abolished their ionophoretic activity. Release of Ca from liposomes which had entrapped arsenazo III-Ca complexes into a medium rich in EGTA permitted calculation of efflux induced by ionophores, whether these were added to the outside of liposomes or preincorporated. Data suggest that phosphatidic acid and oxidized di- and trienoic fatty acids, which act as calcium ionophores in model bilayers, could serve as "endogenous ionophores" in cells.