Phospholipids are normally asymmetrically distributed between leaflets of the plasma membrane, due to the activity of aminophospholipid translocase (APT), a putative plasma membrane Mg2(+)-ATPase which is thought to selectively transport phosphatidylserine (PS) and other aminophospholipids from outer to inner membrane leaflet. Although several candidate proteins have been proposed to serve this function, positive identification awaits demonstration of their capacity to restore APT activity to a cell line that is deficient in this process. This study describes a simple and rapid protocol for the production and selection of mutant cell lines that are defective in APT activity and suitable for expression cloning of cDNAs coding for candidate APT enzymes. By flow cytometry, we demonstrate the time-dependent uptake of NBD-labeled PS, but not phosphatidylcholine (PC), by the mouse fibroblast cell line SV-T2. This uptake was inhibited by known inhibitors of APT, including o-vanadate and N-ethylmaleimide, and by ATP-depletion. SV-T2 cells were mutagenized with ethyl methanesulfonate, and APT-deficient cells were isolated by fluorescence activated cell sorting using NBD-PS as substrate. From a total of 7.2 x 10(6) cells passed through the flow cytometer, 98 clones exhibited APT activity that was less than 50% of that observed for wild-type SV-T2 cells. One clone which exhibited < or = 25% of that observed for wild-type cells, mutant M2711, was further characterized. The defect in mutant M2711 was specific for NBD-PS, and cellular ATP was unchanged, suggesting that the defect in APT activity was not due to a decrease in cellular ATP levels. Mutant M2711 exhibited a growth pattern indistinguishable from that of wild-type SV-T2 cells, and SV-40 large T antigen, which is needed for efficient episomal replication of plasmids containing the SV40 origin of replication, was unchanged. Finally, transfection of M2711 with cDNAs for marker membrane proteins consistently resulted in the same high level of protein expression as that observed for identically-transfected wild-type SV-T2. Thus, flow cytometry can be used for rapid identification of mutants with defects in phospholipid transport that are suitable for functional reconstitution by transfection with candidate APT cDNAs.