Vitrification of bovine oocytes could become the procedure of choice to preserve gametes of valuable females. Most successful vitrification protocols currently include fetal calf serum (FCS) as a component. The aim of this study was to determine the effectiveness of different macromolecules to replace FCS in vitrification solutions for oocyte cryopreservation. Oocytes were matured as described below for 21 h, at 38.5ºC in 5% CO₂ in air, and then partially denuded with 100 IU/ml of hyaluronidase in holding medium (Hepes-TCM 199 with 20% FCS to prevent zona hardening: HM). Oocytes in groups of 24 were then held in HM before vitrification. All vitrification procedures were carried out at 37 + 2ºC. Sub-groups of 4 oocytes were pre-equilibrated in 100 µl of vitrification solution 1 (Hepes-TCM 199 with 10% DMSO, 10% Ethylene Glycol (EG) and the corresponding macromolecule) for 30 sec. Then oocytes were moved through two 100-µl drops of vitrification solution 2 (Hepes-TCM 199 with 20% DMSO, 20% EG, 0.5 M of galactose and the corresponding macromolecule), loaded onto cryoloops (Nat Biotechnol, 17:1234) and immersed in liquid nitrogen after 25 sec. Oocytes were thawed by plunging the cryoloop into 0.5 M galactose in HM, and then placed in 0.5, 0.25 and 0.125 M galactose in HM, and HM alone, for 3 min each. Oocytes were then placed back in maturation medium to complete 23 h of maturation. Frozen thawed semen from one of three bulls was used for fertilization in a chemically defined medium (J Anim Sci 78:152; F-CDM) at 1 million sperm/ml. Zygotes were vortexed after fertilization, and cultured in CDM-1 for 48 h, and then CDM-2 for 144 h, at 38.5ºC, in 5% CO₂, 5% O₂, 90% N₂. Experiment 1: Two maturation media were used: TCM 199 vs CDM, both with the addition of 10% FCS, 1 mg/ml E₂, 1 mg/ml LH and 15 ng/ml FSH. Macromolecule treatments were: A: no macromolecules; B: 20% FCS; C: 6% BSA; D: 2% BSA; E: 18% Ficoll 70,000; F: 6% Ficoll 70,000; G: non-vitrified control; H: non-vitrified but cumulus partly removed control. Experiment 2: Maturation medium was CDM with the addition of 0.5% FAF-BSA, 1 mg/ml E₂, 1 mg/ml LH, 15 ng/ml FSH and 50 µg/ml of EGF. Macromolecule treatments were: A; B; G; H; I: 20% PVP; J: 6% PVP; K: 1% PVA and L: 0.3% PVA. Cleavage was evaluated at 72 h post fertilization, and blastocyst rate was evaluated on days 7, 8 and 9. Each experiment was replicated 6 times. Data were arcsin transformed and analyzed by ANOVA. In experiment 1, there were no differences between maturation media, so data were pooled. Cleavage rates were not different among treatments (P>0.05) ranging from 63 to 77%. Blastocyst rate (per oocyte) was different only in H vs C and A (P<0.05) A: 9; B: 18; C: 12; D: 21; E: 15; F: 15; G: 26 and H: 34%. In Experiment 2 there were no significant differences in cleavage rates (P>0.05) A: 80; B: 75; I: 70; J: 79; K: 76; L: 72; G: 87; H: 83%, but blastocyst rate was different between H vs L and A, and in G vs L (P<0.05) A: 17; B: 23; I: 21; J: 22; K: 20; L: 10; G: 43; H: 34%. In conclusion, with procedures used, vitrification solutions for bovine oocytes containing no macromolecule, 0.3% PVA and 6% BSA (likely due to high viscosity) were inferior to non-vitrified controls. A wide range of other macromolecules and concentrations were, however, suitable for vitrification of bovine oocytes.