The objective of this study was to characterize the circulating concentrations of estradiol-17β (E-17β) after treatment with different types or doses of estrogens in the absence (Experiment 1) or presence (Experiment 2) of a dominant follicle in lactating cows. In Experiment 1, cows (n = 12) had all follicles > 5 mm removed by ultrasound-guided follicular aspiration every 12 h throughout the blood sampling period. Estrogen treatments started 48 h after the first follicular aspiration. Treatments were: no treatment, E-17β (0.5 mg), or estradiol benzoate (EB, 0.5 mg). Seven days after the end of the first trial, cows were then re-randomized to receive: no treatment, E-17β (1.0 mg), EB (1.0 mg), or estradiol cypionate (ECP, 1.0 mg). In Experiment 1, cows treated with E-17β had greater peak circulating concentrations of E-17β than ECP-treated cows, and EB-treated cows had intermediate concentrations. Similarly, E-17β-treated cows had the shortest intervals from treatment to peak concentrations and from peak until return to nadir; ECP-treated cows had the longest intervals, and EB-treated cows had intermediate intervals. In Experiment 2, circulating E-17β was evaluated near the time of AI (artificial insemination) in cows (n = 24) that received Ovsynch with or without E-17β supplementation 48 h after PGF2α treatment. Treatments were: no treatment, E-17β (0.5 mg), or E-17β (1.0 mg). Cows treated with 1.0 mg E-17β had a shorter time to peak circulating E-17β concentrations and greater maximum concentrations (5.0 h; 18.5 pg/ml) than controls (9.5 h; 5.5 pg/ ml), and cows treated with 0.5 mg E-17β were intermediate (5.5 h; 10.6 pg/ ml). Thus, the presence of a dominant follicle and treatment with differing types of estrogen produce substantial differences in the circulating E-17β profile. In lactating dairy cows, a 1.0 mg dose of E-17β increased circulating E-17β concentrations during Ovsynch without disrupting the normal decline in circulating E-17β after the LH surge.