Estradiol and progesterone have been used to synchronize follicular wave emergence and ovulation in a two-dose prostaglandin (PGF)-based synchronization program (Martínez et al. 2004 Theriogenology 62, 363–372). However, it was observed that some heifers displayed estrus prior to the second PGF, suggesting that premature luteolysis may have occurred. An experiment was designed to determine the effects of dose of estradiol benzoate (EB) and/or progesterone (P) on follicular and luteal dynamics in a two dose PGF-based protocol in beef heifers. In two replicates, beef heifers (n = 28; Simmental, Hereford, and Charolais crosses, 350 to 450 kg) received 500 μg cloprostenol (Schering-Plough Animal Health, Pointe-Claire, PQ, Canada) on Day -7. On Day 0, heifers were randomly allocated to nine treatment groups to receive 0, 1, or 2 mg of EB and 0, 50, or 100 mg of P i.m. in canola oil in a 3 × 3 factorial design. A second PGF treatment was administered on Day 14. Ultrasonography was done once daily from Days -5 to 9, and every 12 h thereafter until ovulation. Blood samples were collected at 12-hour intervals from Day 0 to 5 for estradiol and FSH concentrations, and every 24 h for progesterone. The effects of EB and P and their interaction on corpus luteum (CL), follicles, and hormone profiles were analyzed by analysis of variance, and means were compared by LSD or Tukey's test. All variables were normally distributed (Wilk-Shapiro test and rankit plots). The day of follicular wave at the time of treatment tended to vary among groups (P = 0.08) and the diameter of the dominant follicle also differed (P < 0.05). The interval from treatment to wave emergence was shorter (P < 0.05) in heifers that received 2 mg EB (4.6 ± 0.3 d) than in those that did not receive EB (5.9 ± 0.6 d), while the 1 mg EB group (5.1 ± 0.6 d) was intermediate. The interval to wave emergence in the 2 mg EB group was the least variable (P < 0.05). There was no effect of EB (P = 0.72) on the diameter of the CL at the time of the second PGF, but there was an effect of P treatment (P = 0.01). The variability of the interval from the second PGF to ovulation may have been influenced by treatment group but was statistically not significant (P < 0.1). There was an effect of time (P < 0.01) on plasma progesterone concentrations and a P × time interaction (P = 0.06) can also be assumed. Estradiol concentrations were affected by EB dose, time, and EB × time interaction (all P < 0.01). FSH concentrations were modified by time (P < 0.01). In summary, treatment with EB 7 days after a single injection of PGF affected follicular development, while P treatment at that time appeared to influence CL function.