We examined the effects of a single or 2.5-fold dose of dephereline [a gonadotropin-releasing hormone (GnRH) analogue] as well as the drainage of the smaller follicle at the time of insemination on ovulation in dairy cows with two follicles of pre-ovulatory size in the same ovary. The three study groups included 220 monovular cows (control), 110 non-drained cows with two follicles, and 110 cows with two follicles, of which one was drained. In each group, cows treated with a single dose or 2.5-fold dose of dephereline showed similar results following treatment. Ovulation failure of the non-drained follicle occurred in 29.1% of the drained cows, whereas ovulation occurred in 96% of the non-drained and control cows. Twin pregnancy was recorded in 19.4% of the pregnant non-drained cows with two follicles. In conclusion, the increased dephereline dose did not improve the ovulation rate in any group. Follicular drainage, however, prevented twin pregnancy in cows with two follicles, but also resulted in an increase in the non-drained follicle’s rate of ovulation failure.