The objectives of this experiment were to determine the effects of follicular wave (first or second) on diameter of the dominant follicle, concentrations of progesterone and estradiol and the hepatic enzymes that inactivate them, thickness of the endometrium, and pregnancy rates to AI. Beef heifers ( = 101) and cows ( = 106) were randomly assigned to 1 of 2 treatments: insemination to the first follicular wave (FFW) or insemination to the second follicular wave (SFW). Estrous cycles of females were synchronized to ensure appropriate timing for the treatments. The MIXED procedure of SAS was used for analysis. A similar proportion of females in each treatment responded to presynchronization; however, females in the FFW group ovulated in response to the first injection of GnRH of the CO-Synch protocol more frequently. Only females ( = 94) that properly responded to ovulation synchronization were included in further analyses. Cows in the FFW group tended ( 0.06) to have larger ovulatory follicles 36 h post-PGF of the CO-Synch protocol compared to cows in the SFW group (14.22 ± 0.42 and 11.83 ± 0.49, respectively), whereas heifers were similar between treatment groups. Three d prior to AI, circulating concentrations of progesterone were lesser ( 0.01) in females in the FFW (3.63 ± 0.80 ng/mL) than in the SFW (7.12 ± 0.83 ng/mL), whereas concentrations of estradiol tended ( 0.08) to be greater in those in the FFW (82.72 ± 6.48 pg/mL) than in the SFW (65.55 ± 6.74 pg/mL). Concentrations of cytochrome P450 1A in the liver were lesser ( 0.01) in females in the FFW than those in the SFW (0.68 ± 0.08 vs. 0.96 ± 0.06, respectively). Endometrial thicknesses were similar between treatments but were thicker ( < 0.0001) in cows (9.73 ± 0.24 mm) than heifers (7.22 ± 0.26 mm). When considering all females or only those that were properly presynchronized, pregnancy rates were similar between treatments. However, when evaluating females that ovulated to the assigned follicular wave, there was a treatment by parity interaction ( = 0.04) with heifers in the FFW having a lesser pregnancy rate (25.9%) than heifers in the SFW (72.0%) while cows in both treatment groups were intermediate (45.4% in FFW and 50.0% in SFW). The differences in concentrations of steroids between treatment groups may affect fertility of heifers; however, additional research is necessary.