Poor-quality sperm show reduced capacity to undergo capacitation-induced protein tyrosine phosphorylation and hyperactivation. Given that these deficiencies can be overcome by membrane-permeant stimulators of the cAMP-dependent kinase system, we hypothesize that the main defect underlying these deficiencies resides on the sperm plasma membrane. Spermatozoa from semen samples obtained from 15 consenting healthy donors were separated in 2 subpopulations, L45 (first interface) and L90 (pellet), using a 45:65:90 ISolate gradient centrifugation method. These sperm fractions were studied before and after a 6-hour capacitating incubation for sperm motion parameters (computer-assisted analysis), including hyperactivation, protein tyrosine phosphorylation (immunofluorescence), membrane fluidity (Laurdan fluorescence), and sterol and phospholipid content (high-performance thin-layer chromatography). In summary, data indicate that L45 (poor-motility) spermatozoa present an excess of cholesterol and desmosterol, which impairs the normal increase in membrane fluidity during capacitation and its consequent activation of protein tyrosine phosphorylation and hypermotility. Therefore, a defect in membrane composition and dynamics is underlying human sperm biochemical and functional deficiencies related to inadequate capacitation.