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Ram spermatozoa freed from seminal plasma by a dextran 'swim-up' procedure were incubated with Tween 20 and fractionated into motile (PEG-rich) and stationary (dextran-rich) fractions by centrifugal countercurrent distribution (CCCD) in an aqueous dextran-Ficoll-polyethylene glycol (PEG) two-phase system. Increasing concentrations of Tween 20 resulted in greater amounts of extracted protein and lower cell viability. Addition of bull seminal plasma increased the proportion of live cells, whereas ram seminal plasma increased the proportion of stationary cells. Proteins isolated from each type of seminal plasma restored the CCCD profile of treated spermatozoa to the right, this effect being reduced when proteins were thermally denatured. Bovine serum albumin induced a slight displacement to the left. No restoration of profile was achieved when ram spermatozoa were exposed to proteins from bull seminal plasma in the presence of protein-free ram seminal plasma. Adsorption of seminal plasma proteins by spermatozoa previously stripped of surface proteins by exposure to detergent reversed the detergent effect on motility. The findings are consistent with the concept that ram seminal plasma contains a factor that interferes with protein adsorption on the cell surface and prevents the protective effect of seminal plasma proteins on maintenance of cell viability.