The objectives of the current experiment were to determine the effect of interval from reaching activity threshold to artificial insemination with sex-sorted semen on probability of pregnancy. Jersey cows (n = 500) from a commercial dairy herd were fitted with a rumination/activity SCR collar at approximately at 42 ± 7 d postpartum. Cows were presynchronized with 3 injections of prostaglandin-F2α given 14 d apart starting on d 39 ± 3 postpartum. Cows in estrus, based on tail paint removal, after 50 ± 3 d postpartum were inseminated by 1 of 3 technicians. Only cows inseminated in estrus were used in the experiment. Cows were examined for pregnancy at 31 ± 3 and 66 ± 3 d after insemination. A sub-sample of cows (n = 35) were examined by ultrasound every 8 h after onset of estrus to determine time of ovulation, which was determined by the disappearance of a growing follicle ≥10 mm in diameter. Ovulation was estimated to have occurred between the exam in which it was last recorded and the exam in which it had disappeared. Dichotomous data were analyzed by logistic regression with a model that included interval from onset of estrus to insemination (linear, quadratic, and cubic), parity, yield of energy corrected milk, and insemination technician. Interval from onset of estrus to ovulation was analyzed by ANOVA with a model that included size of the follicle at the first ultrasound exam, number of follicles >10 mm in diameter in the first exam, and yield of energy corrected milk. The mean (±SEM) and median intervals from reaching activity threshold to ovulation were 25.7 ± 1.2 h and 27 h (95% CI = 25, 28.2 h), respectively. Interval from onset of estrus to insemination tended to affect probability of pregnancy at 31 ± 3 and 66 ± 3 d after AI in a quadratic manner. Probability of pregnancy 66 ± 3 d after AI increased from 23% to 44%, when AI occurred between 4 and 29 h, respectively, after cows reached activity threshold. Insemination of lactating dairy cows with sex-sorted semen closer to expected ovulation yields the highest probability of pregnancy.