A major site of cryoinjury during cryopreservation of mammalian oocytes is the plasma membrane. Chilling can irreversibly damage plasma membrane integrity during the lipid phase transition that occurs upon cooling. Membranes containing higher cholesterol concentrations are more fluid at lower temperatures and therefore less sensitive to cooling. The purpose of this study was to determine if cryosurvival of vitrified oocytes could be improved by incubation with cholesterol-loaded methyl-beta-cyclodextrin (CLC) prior to vitrification in the presence or absence of fetal calf serum (FCS), and if cholesterol could enter oocytes through cumulus cells and the zona pellucida. Cumulus-enclosed oocytes incubated with various concentrations (0, 0.75 or 1.5 mg/mL) of CLC in the presence of FCS for 25-45 min prior to vitrification did not result in different rates of development after warming of vitrified oocytes, followed by in vitro fertilization. However, there was an increase (P<0.05) in cleavage and number of eight-cell embryos from oocytes preincubated for 1h with 2mg/mL CLC in a chemically defined system and then handled and vitrified in chemically defined media, in comparison to those not exposed to CLC prior to vitrification or to those handled and vitrified in the presence of FCS (55, 41 and 38% eight-cell embryos, respectively). Fluorescence was seen in cumulus-oocyte complexes (COCs) previously exposed to CLC containing cholesterol labeled with a fluorescent dye; fluorescence was also seen in oocytes after removal of the cumulus cells. Oocytes not exposed to the labeled cholesterol did not fluoresce. Cholesterol from CLC readily entered cumulus cells and oocytes and improved survival in chemically defined vitrification systems.