Accelerated transbilayer movement of plasma membrane phospholipids (PL) upon elevation of Ca2+ in the cytosol plays a central role in the initiation of plasma clotting and in phagocytic clearance of injured or apoptotic cells. We recently identified a human erythrocyte membrane protein that induces rapid transbilayer movement of PL at elevated Ca2+. We also presented evidence that this PL scramblase is expressed in a variety of other cells and tissues where transbilayer movement of plasma membrane PL is promoted by intracellular Ca2+ [Zhou, Q., et al. (1997) J. Biol. Chem. 272, 18240-18244]. We have now cloned murine PL scramblase for comparison with the human polypeptide. Both human and murine PL scramblase are acidic proteins (pI = 4.9) with a predicted inside-outside (type 2) transmembrane segment at the carboxyl-terminus. Whereas human PL scramblase (318 AA) terminates in a short exoplasmic tail, murine PL scramblase (307 AA) terminates in the predicted membrane-inserted segment. The aligned polypeptide sequences reveal 65% overall identity, including near identity through 12 residues of an apparent Ca2+ binding motif (D[A/S]DNFGIQFPLD) spanning codons 273-284 (human) and 271-282 (murine), respectively. This conserved sequence in the cytoplasmic domain of PL scramblase shows similarity to Ca2+-binding loop motifs previously identified in known EF hand structures. Recombinant murine and human PL scramblase were each expressed in Escherichia coli and incorporated into proteoliposomes. Measurement of transbilayer movement of NBD-labeled PL confirmed that both proteins catalyzed Ca2+-dependent PL flip-flop similar to that observed for the action of Ca2+ at the cytoplasmic face of plasma membranes. Mutation of residues within the putative EF hand loop of human PL scramblase resulted in loss of its PL mobilizing function, suggesting that these residues directly participate in the Ca2+-induced active conformation of the polypeptide.