Our objective was to assess the effect of treatment with GnRH or 4 increasing doses of human chorionic gonadotropin (hCG) on the ovulatory response of a first-wave dominant follicle and subsequent plasma progesterone (P4) concentrations. Lactating Holstein cows were blocked by parity (primiparous vs. multiparous) and randomly assigned to receive no treatment (control, CON; n = 147), 100 μg of GnRH (n = 144), or 1,000 (n = 138), 2,000 (n = 144), 2,500 (n = 142), or 3,300 (n = 139) IU of hCG 7 d after the last GnRH treatment (G2) of a Double-Ovsynch (DO) or Resynch protocol. Blood samples were collected and ovaries were evaluated with transrectal ultrasonography immediately before treatment and 7 d later to assess serum P4 concentrations and ovulatory response to treatment. Data were analyzed using the MIXED and GLIMMIX procedures of SAS (SAS Institute Inc., Cary, NC). Overall, ovulatory response differed and was 4.8, 79.0, 77.4, 88.9, 92.9, and 95.6% for CON, GnRH, 1,000-, 2,000-, 2,500-, and 3,300-IU hCG treatments, respectively. The increase in plasma P4 concentrations from 7 to 14 d after G2 differed among treatments and was 3.5, 5.9, 5.7, 6.6, 7.0, and 6.5 ng/mL for CON, GnRH, 1,000-, 2,000-, 2,500-, and 3,300-IU hCG treatments, respectively. In conclusion, lactating Holstein cows treated 7 d after G2 with 100 μg of GnRH or 1,000 IU of hCG had similar ovulatory responses (~78%), whereas cows treated with 2,000, 2,500, or 3,300 IU of hCG had increased ovulatory responses (~92%). Ovulatory response of cows treated with 2,000 or 2,500 IU of hCG did not differ, whereas the ovulatory response after 3,300 IU was greater than that after 2,000 IU of hCG. Plasma P4 concentrations and luteal volume 7 d after treatment were increased compared with those of untreated control cows.