The molecular basis of transbilayer movement or flipping of phospholipids in the endoplasmic reticulum is largely unknown. To circumvent the problems inherent to studies with artificial phospholipid analogs, we studied microsomal flip-flop of endogenous phosphatidylethanolamine in yeast. The transbilayer transport of phosphatidylethanolamine was measured in reconstituted proteoliposomes derived from microsomal detergent extracts. Our results demonstrate that flipping is protease sensitive but does not require metabolic energy. Our assay is the first to use the endogenous substrate of the so-called 'flippase' to study phospholipid translocation in endomembranes and may therefore be crucial for the understanding of the catalytic properties of this elusive enzyme.