Aminophospholipid movements in the plasma membrane of higher eukaryotic cells seem to be regulated by an ATP-dependent, protein-mediated process. To examine whether similar mechanisms exist in yeast cells, we have analysed phosphatidylethanolamine (PtdEtn) distributions in Saccharomyces cerevisiae (A184D) cells under a variety of conditions, with trinitrobenzenesulfonic acid and fluorescamine as the external membrane probes. The levels of external PtdEtn in the intact cells were reduced to about 50% by pretreatment of the cells with inhibitors of mitochondrial ATP synthesis, ATPase inhibitors or protein-sulfhydryl-group-modifying reagents, or by depletion of the cells of ATP by metabolic starvation. The levels of external PtdEtn could be restored to normal by repletion of the energy-depleted cells with ATP. Furthermore, treatment of the energy-depleted cells with sulfhydryl-modifying reagents did not cause further reduction in the external PtdEtn levels but decreased the accessibility of PtdEtn to fluorescamine after restoration of the cellular ATP levels to normal in these cells. These results demonstrate an involvement of an ATP-dependent, protein-mediated process(es) in the regulation of the PtdEtn distribution across the plasma-membrane bilayer of yeast cells. The results are discussed with regard to possible models that can generate and maintain the transbilayer phospholipid asymmetry in the yeast plasma membrane.