The hypothesis was tested that estradiol (E2) from the ovarian follicles controls time of luteolysis. Time of luteolysis was evaluated by multiple measures of corpus luteum (CL) structure (area, volume) and function (progesterone [P4], luteal blood flow). The hypothesis for experiment 1 was that repeated ablation of follicles would reduce circulating E2 and delay luteolysis. Heifers were randomly assigned on Day 9 (Day 0 = ovulation) to three groups. All follicles ≥4 mm were ablated on Day 9 (group FA9; n = 6); Days 9–15 (group FA15; n = 6); or Days 9–21 (group FA21; n = 7). As expected, follicular ablation delayed (P < 0.001) the rise in circulating E2 and peak E2 concentrations (FA9, Day 17.6 ± 0.7; FA15, Day 20.3 ± 0.3; FA21, Day 24.9 ± 0.3). Luteolysis (based on each measure) was delayed (P < 0.005) by repeated ablation of follicles, with earlier luteolysis (based on P4 decrease) in FA9 (Day 15.2 ± 0.8) than FA15 (Day 16.5 ± 0.4), and a further delay in FA21 (Day 18.3 ± 0.5). The hypothesis of experiment 2 was that exogenous treatment with E2 would stimulate prostaglandin F_2alpha (PGF) secretion and prevent the delay in luteolysis associated with follicular ablations. Follicles ≥4 mm were ablated from Day 9 to Day 17 (n = 15). Heifers were treated on Days 13 and 15 with 1.0 mg of estradiol benzoate (FAE2; n = 7) or vehicle (FAV; n = 8). Treatment with E2 induced PGF secretion (detected by PGF metabolite) and induced earlier (P < 0.02) luteolysis in FAE2 than in FAV, whether determined by circulating P4 or by area, volume, or blood flow of CL. In summary, ablation of follicles (≥4 mm) delayed and treatment with E2 hastened luteolysis in heifers with ablated follicles. Thus, these results are consistent with an essential role for follicle E2 in timing of luteolysis.