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The objective of the current study was to evaluate the effect of lactation on oocyte quality and in vitro embryo production in Gyr (Bos indicus) cows. A total of 187 multiparous cows (92 lactating and 95 non-lactating) were subjected to a single session of follicular aspiration. Follicle puncture was performed on a random day of the estrous cycle, without prior application of hormonal drugs. The recovered oocytes were subjected to maturation, fertilisation, and in vitro culture. The results indicated that in lactating Gyr cows had the following values of studied variables: number of follicles visualised (22.1±11.38), number of recovered oocytes (17.4±10.21), number of grade III oocytes (7.0±5.86), rate of viable oocytes (72.1±13.11%), number of blastocysts D7 (2.5±2.26) and blastocyst rate (18.3±15.61%), in relation to non-lactating cows (27.7±11.53; 21.1±10.06; 9.8±6.08, 77.4±10.08%; 4.9±3.34; 30.2±15.41%, respectively). In conclusion, the lactation period in Gyr cows affects the performance of in vitro embryo production programmes. [ABSTRACT FROM AUTHOR]