Despite enormous interest in membrane raft micro‐domains, no studies in any cell type have defined the relative compositions of the raft fractions on the basis of their major components—sterols, phospholipids, and proteins—or additional raft‐associating lipids such as the ganglioside, G_M1. Our previous localization data in live sperm showed that the plasma membrane overlying the acrosome represents a stabilized platform enriched in G_M1 and sterols. These findings, along with the physiological requirement for sterol efflux for sperm to function, prompted us to characterize sperm membrane fractions biochemically. After confirming limitations of commonly used detergent‐based approaches, we utilized a non‐detergent‐based method, separating membrane fractions that were reproducibly distinct based on sterol, G_M1, phospholipid, and protein compositions (both mass amounts and molar ratios). Based on fraction buoyancy and biochemical composition, we identified at least three highly reproducible sub‐types of membrane raft. Electron microscopy revealed that raft fractions were free of visible contaminants and were separated by buoyancy rather than morphology. Quantitative proteomic comparisons and fluorescence localization of lipids suggested that different organelles contributed differentially to individual raft sub‐types, but that multiple membrane micro‐domain sub‐types could exist within individual domains. This has important implications for scaffolding functions broadly associated with rafts. Most importantly, we show that the common practice of characterizing membrane domains as either “raft” or “non‐raft” oversimplifies the actual biochemical complexity of cellular membranes. J. Cell. Physiol. 218: 537–548, 2009. © 2008 Wiley‐Liss, Inc.