During the maturation process spermatozoa undergo a series of changes in their lateral and horizontal lipid profiles. However, lipid metabolism in spermatozoa is not clearly understood for two reasons: i) the mature spermatozoa are devoid of endoplasmic reticulum, which is the major site of phospholipid (PL) synthesis in somatic cells, and ii) studies have been superficial due to the difficulty in culturing spermatozoa. We hypothesize that spermatozoa contain biogenic membrane flippases since immense changes in lipids occur during spermatogenic differentiation. To test this, we isolated spermatozoa from bovine epididymides and reconstituted the detergent extract of sperm membranes into proteoliposomes. In vitro assays showed that proteoliposomes reconstituted with sperm membrane proteins exhibit ATP-independent flip-flop movement of phosphatidylcholine (PC), phosphatidylserine, and phosphatidylglycerol. Half-life time of PC flipping was found to be ∼3.2±1 min for whole sperm membrane, which otherwise would have taken ∼11-12 h in the absence of protein. Further biochemical studies confirm the flip-flop movement to be protein-mediated, based on its sensitivity to protease and protein-modifying reagents. To further determine the cellular localization of flippases, we isolated mitochondria of spermatozoa and checked for ATP-independent flippase activity. Interestingly, mitochondrial membranes showed flip-flop movement but were specific for PC with half-life time of ∼5±2 min. Our results also suggest that spermatozoa have different populations of flippases and that their localization within the cellular compartments depends on the type of PL synthesis. © 2013 Society for Reproduction and Fertility.