Cryopreservation of cytoplasts would help to resolve the logistics of matching the \navailability of oocytes with embryo donors in nuclear transfer. Therefore, the developmental \npotential of nuclear transfer bovine embryos reconstructed using vitrified cytoplasts was \ninvestigated. In vitro matured oocytes were denuded, enucleated, activated with calcium \nionophore (10 p.M, 5 min) and cycloheximide (10 Ixg/mL, 6 h) and then vitrified by the open \npulled straw (OPS) method. After immediate warming, the nuclear transfer embryos were \nreconstructed using blastomeres from nonvitrified, in vitro-produced embryo donors. Compared \nwith control nuclear transfer embryos that were reconstructed using nonvitrified cytoplasts, \nfusionrates (% ± SEM) were not affected (83.7 ± 9.2 vs 79.8 ± 4.6; P>0.05), but cleavage (55.7 ± \n2.9 vs 92.8 ± 3.9; P=0.0002) and blastocyst rates (7.2 ± 5.0 vs 32.6 ± 7.8; P=0.0025, vitrified vs \nnonvitrified cytoplasts, respectively) per successful fusion were reduced. One nuclear transfer \nblastocyst reconstructed from a vitrified cytoplast was transferred to a synchronized recipient. \nAfter a normal length gestation (265 d), twin calves (21 and 26 kg) were delivered. Microsatellite \nanalysis confirmed that the calves were homozygotic (the embryo split in utero), and were \nderived from the in vitro-produced embryo donor. The twins were dead at birth, but post-mortem \nanalysis of the calves indicated no abnormalities or infections, suggesting that their death was \nrelated to the twin pregnancy and the known fragility of nuclear transfer calves. These data \ndemonstrate that open pulled straw-vitrified cytoplasts are capable of supporting full-term \ndevelopment of nucleartransfer embryos.