Our objectives were to identify stages of the estrous cycle at which initiation of a timed artificial insemination (Ovsynch/TAI) protocol may reduce pregnancy rates and to monitor ovarian follicle dynamics and corpus luteum development after initiation of the Ovsynch/TAI protocol at different stages of the cycle. Cycling Holstein heifers (n = 24) were injected twice with prostaglandin F2alpha to induce estrus and were scanned by ovarian ultrasonography to determine the day of ovulation (d 0). Heifers were assigned to initiate the Ovsynch/TAI protocol at d 2 (n = 5), 5 (n = 5), 10 (n = 4), 15 (n = 5), or 18 (n = 5) of the cycle. The Ovsynch/TAI was initiated with an injection of gonadotropin-releasing hormone agonist followed 7 d later with an injection of prostaglandin F2alpha. At 36 h after injection of prostaglandin F2alpha, heifers were injected with gonadotropin-releasing hormone agonist and inseminated 16 h later. Heifers were scanned daily during the Ovsynch/TAI protocol and every other day after insemination until 16 d later. Blood samples were collected daily starting at the 1st day heifers were scanned and continued until 16 d after insemination. Initiation of the Ovsynch/TAI protocol at d 15 of the estrous cycle caused heifers to ovulate prior to insemination. A shortened return to estrus (< 16 d) was caused by ovulation failure to the second gonadotropin-releasing hormone injection, by incomplete regression of the corpus luteum, and by short life-span of the induced corpus luteum. Day of the cycle in which the Ovsynch/TAI protocol is initiated affects dynamics of follicular development, plasma progesterone profiles, and occurrence of premature ovulation. Size of the pre-ovulatory follicle was associated positively with subsequent progesterone concentrations following insemination.