A relatively rapid transbilayer motion of phospholipids in the microsomal membrane seems to be required due to their asymmetric synthesis in the cytoplasmic leaflet. Marked discrepancies exist with regard to the rate and specificity of this flip-flop process. To reinvestigate this problem, we have used both spin-labeled and radioactively labeled long chain phospholipids with a new fast translocation assay. Identical results were obtained with both types of probes. Transbilayer motion of glycerophospholipids was found to be much more rapid than previously reported (half-time less than 25 s) and to occur identically for phosphatidylcholine, phosphatidylserine, and phosphatidylethanolamine. Such transport is nonvectorial and leads to a symmetric transbilayer distribution of phospholipids. In contrast, transverse diffusion of sphingomyelin was 1 order of magnitude slower. Phospholipid flip-flop appears to occur by a protein-mediated transport process displaying saturable and competitive behavior. Proteolysis, chemical modification, and competition experiments suggest that this transport process may be related to that previously described in the endoplasmic reticulum for short-chain phosphatidylcholine (Bishop, W. R., and Bell, R. M. (1985) Cell 42, 51-60). The relationship between phospholipid flip-flop and nonbilayer structures occurring in the endoplasmic reticulum was also investigated by 31P-NMR. Several conditions were found under which the 31P isotropic NMR signal previously attributed to nonbilayer structures is decreased or abolished, whereas transbilayer diffusion is unaffected, suggesting that the flip-flop process is independent of such structures. It is concluded that flip-flop in the endoplasmic reticulum is mediated by a bidirectional protein transporter with a high efficiency for glycerophospholipids and a low efficiency for sphingomyelin. In vivo, the activity of this transporter would be able to redistribute all changes in phospholipid composition due to biosynthetic processes between the two leaflets of the endoplasmic reticulum membranes within a time scale of seconds.