The COSynch protocol has been used to synchronize ovulation and facilitate fixed-time AI in beef cattle. Establishment and maintenance of pregnancy was negatively affected, in previous studies, by GnRH-induced ovulation of small dominant follicles (</=11 mm). The reason for the presence of small follicles at the second GnRH (GnRH 2) is not clear. The objectives of this study were 1) to determine the effect of ovulatory response at the first GnRH (GnRH 1) on diameter and variation in diameter of the largest follicle at GnRH 2, and 2) to determine the effect of day of the cycle (stage of a follicular wave) on GnRH-induced luteinizing hormone (LH) release, and the resulting ovulatory response after GnRH 1 and 2. Two experiments used pubertal beef heifers synchronized to be on different days of the estrous cycle (d 2, 5, 10, 15, and 18 after estrus) in which a dominant follicle would or would not respond to GnRH 1. Ovulatory response to GnRH 1 did not affect size or variation in diameter of the largest follicle at GnRH 2 in Exp. 1 or 2. In Exp. 1, ovulatory response after GnRH 1 (0/14(a), 12/13(b), 4/13(ac), 9/13(bc), and 2/10(a) in the d 2, 5, 10, 15, and 18 groups; (a-c)P < 0.05) and GnRH 2 (13/14(a), 12/13(a), 12/13(a), 2/13(b), and 2/10(b) in the d 2, 5, 10, 15, and 18 groups, respectively; (a,b)P < 0.05) was affected by day of the cycle. In Exp. 2, day of the cycle also affected the proportion of heifers ovulating after GnRH 1 (0/7(a), 8/8(b), 0/6(a) 5/8(ab), and 5/8(ab) of the d 2, 5, 10, 15, and 18 heifers, respectively; (a-c)P < 0.05) and GnRH 2 (3/7(ab), 8/8(b), 5/6(b), 1/8(a), and 2/8(a) of the d 2, 5, 10, 15, and 18 heifers, respectively; (a,b)P < 0.05). In both experiments, heifers receiving GnRH 1 on d 15 and 18 had a greater (P < 0.05) occurrence of luteolysis before PGF(2alpha) injection and expression of estrus than heifers treated on d 2, 5, and 10. The GnRH-induced LH surge was of greatest magnitude in heifers receiving GnRH 1 on d 18 of the cycle followed by d 5, 15, 10, and 2 (9,054(b), 5,774(bc), 4,672(c), 2,548(c), and 915(d) arbitrary units; respectively; (a-d)P < 0.05). In summary, ovulatory response to GnRH 1 did not affect size of the dominant follicle at GnRH 2. Day of the cycle when GnRH 1 was delivered affected dominant follicle size at GnRH 2. Treatment with GnRH 1 in the earlier part of the estrous cycle (on or before d 10) increased the proportion of dominant follicles that were large enough to respond to GnRH 2 (>/=10 mm) and increased ovulatory response after GnRH 2.