The objectives of this experiment were to determine 1) the effect of a single injection of porcine follicle stimulating hormone (FSHp) on Day 1 of the estrous cycle on the number of estrogen-active (EA dominant) follicles present on Days 3, 5 and 7 of the same cycle, and 2) the relationship between concentrations of estradiol and inhibin in these follicles. Cyclic beef heifers (n = 38) were treated with a synthetic analogue of prostaglandin F2-alpha (Luprostiol, 15 mg) to synchronize estrus (Day 0). On Day 1, heifers were randomly assigned to one of two treatments: 7 mg FSHp (Folltropin, n = 18); or controls (n = 20). Heifers were slaughtered on Days 3, 5 or 7 and the number of follicles greater-than-or-equal-to 5mm was recorded. Follicular fluid from each follicle greater-than-or-equal-to 5mm was aspirated, quantified, centrifuged and frozen for subsequent determination of estradiol 17-beta (E2), progesterone (P4) and inhibin concentrations. Follicles were classified as estrogen-active if the concentration of estradiol 17-beta exceeded that of progesterone in follicular fluid. A single injection of FSHp on Day 1 had no significant effect (P > 0.05) on the overall number of follicles on Days 3, 5 and 7; FSHp treatment possibly increased the number of large follicles on Day 3 (P = 0.04). There were more estrogen-active follicles on Day 3 than on Day 5 or Day 7 irrespective of treatment. The FSHp treatment had a significant effect on the proportion of estrogen-active follicles when all follicles were considered together (94% estrogen-active for control, 50% for FSHp treatments; P = 0.025) or when only medium follicles were assessed (92% for control, 28% for FSHp treatments; P = 0.0001); this effect was not significant when only the large follicles were considered. No significant correlations were found between concentrations of inhibin, estrogen or progesterone in medium or large follicles. In conclusion, a single injection of FSHp on Day 1 did not significantly affect the total number of follicles, and it possibly increased the number of large follicles on Day 3 and decreased the percentage of estrogen-active follicles on Day 3.