Here, ESR and membrane anisotropy techniques were used to collect informations on sperm membrane fluidity. It should be noted that both R428 Axl inhibitor methods used probes that bind membrane lipids in an unpredictable manner. Therefore it could be hypothesized that the binding properties of both the probes may be strictly dependent of sperm membrane composition and, as a consequence, of sperm functional state. For this reason, S or r parameters were compared and used to obtain a mean value of membrane fluidity, that may be the expression of the different membrane districts and of different sperm cell subpopulations. For this reason EPR and membrane anisotropy were integrated with KRX-0401 filipin staining, a reliable qualitative approach yielding information on the incidence of different sperm subpopulations before and after cholesterol extraction. On the basis of these results obtained with different techniques, the reduction of S and r parameters following cholesterol extraction induced by MCD could be easily interpreted. Filipin measurements indicated, in fact, that this cyclic oligosaccharide was able to extract cholesterol from sperm membranes independently of the capacitation status and, therefore, of the preliminary disorder in phospholipid transmembrane distribution. On the contrary, BSA exposure was influenced by the priming effect of bic on phospholipid organization. Its ability to extract cholesterol, as indicated by combining P and r parameters, was inhibited in Met-AEA treated cells, when a lower incidence of pattern C of filipin fluorescence was recorded. In addition, the effect of cholesterol extraction from sperm membrane seems to confirm what observed in cellular models where cholesterol influence on membrane fluidity was correlated to the surrounding molecules. Cholesterol extraction decreases membrane fluidity, when it is close to unsaturated fatty acid; instead, if saturated fatty acids are more abundant, cholesterol acts by promoting an increase in membrane fluidity and, as a consequence, by lowering membrane order. The boar spermatozoa membrane contains relatively high amounts of unsaturated phospholipids and, for this reason, cholesterol extraction induced by BSA or MCD reduced both S and r parameters. Taken together, converging evidence supports the unprecedented concept that AEA can inhibit capacitation by preventing an increase in membrane fluidity, that is recognized as a key parameter involved in the acquisition of the fertilizing ability of spermatozoa. In fact, during capacitation the plasma membrane and the outer acrosome membrane become less stable and gradually acquire the ability to fuse with each other.