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Abstract: Circulating concentrations of hormones were determined each hour in 13 heifers from the end of the luteolytic period to ovulation (follicular phase, 3.5 days). Diameter of the preovulatory follicle was determined every 8 hours, and the time of ovulation was determined hourly. The diameter of the preovulatory follicle decreased 0.8 ± 0.1 mm/h in heifers when there was 1 to 3 hours between the last two diameter measurements before ovulation. The concentration of progesterone (P4) after the end of the luteolytic period (P4 < 1 ng/mL) changed (P < 0.0001), as shown by a continued decrease until Hour −57 (Hour 0 = ovulation), then was maintained at approximately 0.2 ng/mL until 2 hours before the peak of the LH surge at Hour −26, and then a decrease to 0.1 ng/mL along with a decrease in estradiol-17β. Concentrations of LH gradually increased (P < 0.007) and concentrations of FSH gradually decreased (P < 0.0001) after the end of luteolysis until the beginning nadirs of the respective preovulatory surges. A cluster of prolactin (PRL) pulses occurred (P < 0.0001) each day with approximately 24 hours between the maximum value of successive clusters. Hourly concentrations of a PGF2α metabolite decreased (P < 0.007) until Hour −40, but did not differ among hours thereafter. Novel observations included the gradual increase in LH and decrease in FSH until the beginning of the preovulatory surges and follicle diameter decrease a few hours before ovulation. Results supported the following hypotheses: (1) change in the low circulating P4 concentrations during the follicular phase are temporally associated with change in LH concentrations; and (2) PRL pulses occur in a cluster each day during the follicular phase of the estrous cycle. [Copyright &y& Elsevier]