Transvaginal ultrasound-guided follicle aspiration (TVA) is the most effective way to recover multiple immature oocytes from live mares. Because of the tight attachment of the equine immature oocyte to the follicle wall, TVA in this species is time consuming, taking up to 1 h per horse. Thus, it may be difficult to search follicular aspirates immediately after recovery. In 2009 in a series of 6 replicates, we observed a blastocyst development rate of 32% (13/41) after intracytoplasmic sperm injection of oocytes collected by TVA and held for ~1.5 h at ambient temperature (26 to 32°C) before isolation from aspirated fluid (unpublished data). Therefore, in the present study we compared the effects of immediate oocyte isolation v. holding the follicular aspirate before oocyte isolation on oocyte maturation and blastocyst rates. Ten mares were used for this study; TVA was performed on each mare every 14 days, for 4 aspiration sessions per mare. Collected aspiration fluid was either processed immediately or held for 2 h at 32°C before processing. At each aspiration, one ovary was randomly assigned to each treatment [immediate (Imm) or 2-h holding (2-H)]. Follicle aspiration was performed as previously described (Jacobson et al. 2010 Theriogenology 73, 1116–1126) using M199 with Hank’s salts to flush the follicle lumen up to 8 times per follicle. Oocytes were recovered from the aspirates by filtration. Overall, 325 follicles were aspirated and 140 oocytes were obtained (43% recovery). The proportion of degenerating oocytes was not significantly different between treatments (1/68 and 0/72 for Imm and 2-H, respectively). Oocytes were held overnight in modified M199 as previously described (Choi et al. 2006 Theriogenology 66, 955–963) before maturation culture. After 30 h of maturation culture, there was no significant difference in maturation rates between treatments [75% (50/67) and 65% (46/71) for Imm and 2-H, respectively]. Fertilization was performed by intracytoplasmic sperm injection, and injected oocytes were cultured 7 to 10 days, as previously described (Choi et al. 2006). The rates of blastocyst development per injected oocyte were 23% (11/47) for Imm and 16% (7/44) for 2-H; these were not significantly different (P > 0.3). The reason for the discrepancy in blastocyst rates for held oocytes in this study (16%) compared with our 2009 observations (32%) is unclear; this could be a factor of the time held (~1.5 v. 2 h), the temperature (26–32°C v. 32°C), or minor changes in protocol between the 2 years. From these results, we conclude that holding oocytes in the follicular aspirate for up to 2 h following collection may be performed when necessary without significantly affecting the rate of subsequent blastocyst development.