The objective was to investigate the effect of presynchronization with PGF prior to a Cosynch protocol on estrus synchrony, CL and preovulatory follicle diameters and pregnancy rate following timed-AI (TAI) in beef heifers. Cycling beef heifers (n = 148) were treated with 100 μg GnRH i.m. (Cystorelin, Merial Canada Inc., Victoriaville, Quebec, Canada) on Day 0, 500 μg cloprostenol i.m. (PGF; Estrumate, Schering Plough Animal Health, Pointe-Claire, Quebec, Canada) on Day 7, and GnRH concurrent with TAI on Day 9 (54 h after PGF). Half of the heifers (Control) received the first GnRH treatment at random stages of the estrous cycle, while the other half (Presynch) received two injections of PGF 11 days apart, with the first injection of GnRH 11 days after the second injection of PGF. Estrus detection was done between the first GnRH and 12 h after PGF, and heifers detected in estrus were inseminated (and considered nonpregnant to TAI), while all other heifers were TAI. Heifers were examined by transrectal ultrasonography for CL and follicle development, and confirmation of pregnancy. Data were compared between groups using Student’s t-test and chi-square procedures. The numbers of heifers in estrus early (after first GnRH and before TAI) was higher in the Control group than in the Presynch group (18/74 v. 2/74, respectively; P < 0.0001). Mean (±SD) diameters of the dominant follicle (12.1 ± 3.1 v. 14.2 ± 2.5 mm) and CL (17.3 ± 5.5 v. 20.5 ± 4.3 mm) at first GnRH injection were smaller (P < 0.0001) and more variable (P < 0.03) in Control than Presynch heifers, but diameters of the preovulatory follicle (P = 0.3) and CL (P = 0.1) at TAI did not differ. Although the diameter of the preovulatory follicle was more variable (P < 0.004) in Control (5 to 19 mm) than Presynch (8 to 17 mm) heifers, pregnancy rate to TAI did not differ (P = 0.4; 29.7 v. 36.5%, respectively). Overall pregnancy rates were 45.9 and 37.8% for Control and Presynch groups, respectively (P = 0.3). Pregnancy rate tended (P < 0.08) to be affected by diameter of the preovulatory follicle at the time of TAI (0, 23.1, 45.7, 41.4, and 60.0% pregnant for diameters of < 9, 9–11, 12–14, 15–17, and > 17 mm, respectively). Regardless of treatment, diameter of the preovulatory dominant follicle (P < 0.02) and CL (P < 0.03) 7 days after TAI was smaller, and CL diameter was more variable (P < 0.004), in open than in pregnant heifers (12.7 ± 2.6 v. 13.8 ± 2.1 mm, and 16.5 ± 4.4 v. 18.0 ± 3.0 mm, respectively). In summary, presynchronization with PGF prior to a Cosynch protocol reduced the proportion of heifers in estrus before TAI, suggesting that this approach may be useful in the successful application of Ovsynch or Cosynch programs in heifers. However, pregnancy rate to TAI did not differ between groups in this study. Diameter of the preovulatory follicle tended to positively affect pregnancy rate, regardless of treatment.