Treatment with GnRH in a controlled internal drug release (CIDR)-based timed AI protocol induced synchronized follicular wave emergence, a large ovulatory follicle, and synchronous ovulation following a second injection of GnRH, while follicular wave emergence was relatively asynchronous in the estradiol benzoate (EB)-treated cows resulting in ovulation of smaller ovulatory follicles (Kim et al. 2004 Theriogenology, in press). In this study, we compared new follicular wave emergence and follicular development following treatment with EB plus progesterone (P4) or GnRH at the first follicular wave in CIDR-treated, lactating Holstein cows. Previously synchronized, lactating Holstein cows (n = 20) received a CIDR (CIDR^™, InterAg, Hamilton, New Zealand, Day 0) 3 to 8 days after ovulation and were randomly assigned to two treatments: cows in the GnRH group (n = 10) received 100 μg fertirelin acetate i.m. (GnRH, Conceral, Dongbang Co., Seoul, Korea) and those in the E/P group (n = 10) received 2 mg EB (SY Esrone, Samyang, Seoul, Korea) and 50 mg P4 i.m. (SY Ovaron, Samyang, Seoul, Korea) at that time. Thereafter, all animals received PGF_2α (Lutalyse, Pharmacia & Upjohn, Puurs, Belgium) at the time of CIDR removal on Day 7, and GnRH on Day 9. Ovaries of each cow were examined by transrectal ultrasonography (Sonoace 600 with 5.0 MHz linear-array transducer; Medison Co., Ltd., Seoul, Korea) every 24 h from Days 0 to 9, on Day 11, and on Day 14 of treatment to observe changes in ovarian structures (follicles and CL). Data were analyzed by Fisher's exact test, Wilcoxon Rank-Sum test and Student's t-test using an SAS program. Follicular wave emergence occurred within 7 days in 9/10 GnRH- and 8/10 E/P-treated cows (P > 0.05). The interval to wave emergence was longer (P < 0.01) in the E/P group (5.9 ± 0.5 days) than in the GnRH group (2.9 ± 0.4 days). While diameters of dominant follicles on Day 0 were not different (P > 0.05) between groups, diameters on Day 7 and Day 9 in the E/P group (9.4 ± 0.4 and 13.1 ± 0.7 mm, respectively) were significantly smaller (P < 0.01) than those in the GnRH group (12.9 ± 0.5 and 15.8 ± 0.7 mm, respectively). Growth rates of preovulatory follicles in the E/P group (2.1 ± 0.2 mm/day) were greater (P < 0.01) than in the GnRH group (1.2 ± 0.1 mm/day), while the duration of dominance of the preovulatory follicle was longer (P < 0.01) in the GnRH group (6.3 ± 0.4 days) than in the E/P group (3.3 ± 0.5 days). Synchronized ovulation by 40 h after the GnRH injection (Day 11) occurred in 10/10 GnRH- and 9/10 E/P-treated cows (P > 0.05). Results suggest that estradiol benzoate plus progesterone administered at the first follicular wave in CIDR-treated, lactating Holstein cows delays new follicular wave emergence, resulting in ovulation of smaller ovulatory follicles than GnRH. Endocrine changes during treatment and fertility following timed AI has been investigated in this study.