The relationship between multiple ovulations and various factors involved in conception in lactating Holstein cows was investigated in this study. A total of 53 AI cows housed in a free-stall barn were assigned to this study. They were offered a balanced TMR ad libitum during the experimental period. Cows were milked at 0830 and 1900 h. Milk yields were recorded at each milking. Estrus was detected by radiotelemetric pedometers (Yoshioka et al. 2007 Reprod. Fertil. Dev. 19, 129). The body condition score (BCS) of cows was recorded at estrus. Artificial insemination was performed according to the pedometry data. Pregnancy was diagnosed 42 days post-AI by transrectal ultrasonography. Ovulation was assessed by transrectal ultrasonography of the ovary every 3 h from the end of estrus. Ovulation time was measured from the onset of estrus to ovulation. Milk production of each cow was determined from 14 days before the day of estrus. Blood samples for estradiol-17β (E2) were collected at the peak of estrus, and for progesterone (P4), samples were collected on Days 0, 3, 6, 9, and 12 (estrus = Day 0). The E2 and P4 concentrations were measured by enzyme immunoassay. Preovulatory follicles at estrus and CL on Day 0, 3, 6, 9, and 12 were monitored by transrectal ultrasonography. The overall incidence of multiple ovulations was 28.3% (15/53). There was no difference in duration of estrus between cows with multiple ovulations and cows with single ovulations (10.2 h, n = 15 v. 9.8 h, n = 38). Ovulation time was longer for cows with multiple ovulations than that for cows with single ovulations (30.4 h, n = 10 v. 28.7 h, n = 26; P = 0.0545). In all cases of multiple ovulations, 2 follicles ovulated simultaneously. The milk production of cows with multiple ovulations was higher than that of cows with single ovulations (40.1, n = 15 v. 36.2 kg d^–1, n = 38; P = 0.0515); however, BCS showed the opposite results (2.86, n = 15 v. 3.13, n = 38; P < 0.05). There was a significant inverse correlation between milk production and BCS in cows with multiple ovulations (r = –0.768; P < 0.01). The E2 concentrations showed no difference between cows with multiple and single ovulations (9.6 pg mL^–1, n = 8 v. 8.2 pg mL^–1, n = 23). The diameters of preovulatory dominant follicles and secondary follicles at estrus in cows with multiple ovulations were significantly smaller than those in cows with single ovulations (18.2 and 14.2 mm, n = 14 v. 19.9 mm, n = 38, respectively; P < 0.05). Cows with multiple ovulations showed a significantly higher P4 concentration and a larger CL than those with single ovulations from Day 3 to 12. The conception rate for AI was significantly higher for cows with multiple ovulations than for cows with single ovulations (73.3%, n = 15 v. 36.2%, n = 38; P < 0.05). Our results showed that high conception rates, high levels of milk production, and low BCS were associated with multiple ovulations.