We tested the hypothesis that a small dose of estradiol benzoate (EB) at the midstage of the estrous cycle in cattle would synchronize the subsequent pattern of ovarian follicular development, estrus, and ovulation. Nonlactating Friesian cows received either 1 mg of EB i.m. on d 13 of the estrous cycle (T; n = 12; estrus = d0) or served as untreated controls (C; n = 12). Their ovaries were examined daily with transrectal ultrasonography from d 7, and blood samples were collected 0, 2, 4, 8, 24, and 48 h after treatment on d 13. Plasma concentrations of estradiol-17beta were elevated to 12 pg/mL during the initial 24 h following treatment, compared with a baseline of 1 pg/mL in untreated controls (P < .001). Progesterone concentrations in cows of the T group declined between 24 and 48 h after treatment (-3.2 +/- .5 ng/mL) compared with little change in concentrations of progesterone in cows of the C group at this time (P < .01). This difference was coincident with an earlier time to regression of the corpus luteum in cows of the T group. Disregarding treatment groups, the second dominant follicle of the estrous cycle (DF2) emerged on d 10.6 +/- .3 and was 9.4 +/- .4 mm in diameter on d 13. Further growth of the DF2 was halted by EB treatment on d 13. Cessation of growth occurred irrespective of whether the DF2 was in the early or late growth phase, and a new follicular wave emerged 4.5 +/- .2 d later. The dominant follicle from this wave (DF3) ovulated 5 d after emergence in most cases. During the estrous cycle of every cow in the T group, there were three waves of follicular development (3-wave), whereas the ratio of 2:3 waves of follicular development in cows of the C group was 1:3. Consequently, the interval from emergence to ovulation of the ovulatory dominant follicle in cows of the C group ranged from 3 to 11 d. The dynamics of ovarian follicular wave development during the estrous cycle can be strategically manipulated by treating with a small dose of EB to synchronize proestrous development of the ovulatory follicle.