Sperm membranes suffer significant lipid changes during cryopreservation similar to initial steps in capacitation, in which a reduction in plasma membrane cholesterol (pmCHO) is observed. Methyl-β-cyclodextrin (MBCD) or cyclodextrin pre-loaded with cholesterol (CLC; Purdy PH and Graham JK 2004 Cryobiology 48, 36–45) have been used to decrease or increase the pmCHO, respectively, in different mammalian spermatozoa. In this study, pmCHO levels were assessed during the cryopreservation process in boar sperm samples deprived of (D) or enriched in (E) CHO. Single sperm-rich fractions from 14 boars were divided in 4 aliquots and frozen in 0.5-mL straws after dilution in a lactose-egg yolk extender with a final concentration of 20% egg yolk and 3% glycerol (Control sample, C) and supplemented with MBCD (1 mg/120 × 10⁶ cells; D sample) or CLC at 1 (E-1 sample) or 3 (E-3 sample) mg/120 × 10⁶ cells. The pmCHO level was quantified at 17°C in pre-diluted sperm samples (basal pmCHO) and at 3 steps of the cryopreservation process: after cooling at 5°C (step 1), after the addition of 3% glycerol (step 2), and immediately after thawing (step 3). The pmCHO was quantified by an enzymatic colorimetric test (Spinreact^®, Sant Esteve de Bas, Spain) at 520 nm, following the protocol described by Moore AI et al. (2005 Cryobiology 51, 241–249). Data (least squares means ± SEM) were expressed as micrograms of CHO/10⁶ sperm and analyzed as a mixed-model ANOVA. Because there were significant differences (P ≤ 0.05) between ejaculates, this effect was included as random. The level of pmCHO in C, D, and E-1 samples followed the same trend without significant differences (P ≤ 0.05) among them. That level increased (P ≤ 0.05) at step 1 (0.55 ± 0.17; 0.52 ± 0.17; and 0.66 ± 0.17 for C, D, and E-1, respectively), compared with the basal level (0.24 ± 0.02), and decreased (P ≤ 0.05) to the basal level at steps 2 (0.23 ± 0.07; 0.23 ± 0.07; and 0.30 ± 0.07 for C, D, and E-1, respectively), and 3 (0.07 ± 0.02; 0.08 ± 0.02; and 0.13 ± 0.02 for C, D, and E-1, respectively). Although the pattern in E-3 was similar to other treatments, the level of pmCHO was greater (P ≤ 0.05) than those at step 1 (0.75 ± 0.17), 2 (0.51 ± 0.07), and 3 (0.16 ± 0.02). In conclusion, the pre-freezing treatment of sperm samples with methyl-β-cyclodextrin, to reduce the CHO, did not modify the CHO level of plasma membrane of boar spermatozoa. However, treatment with cyclodextrin pre-loaded with CHO at 3 mg/120 × 10⁶ cells increased significantly the CHO level of plasma membrane, which was evident throughout the cryopreservation process.