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Recent studies have implicated insulin-like growth factor I (IGF-I) as an intraovarian regulator of follicular growth and differentiation. Therefore, we investigated the possibility that cattle selected for twin births may have increased concentrations of IGF-I within the ovarian follicle and(or) in peripheral blood. The estrous cycles of 14 cows with histories of producing twins and 12 control monotocous cows were synchronized with 35 mg of prostaglandin F2α (PGF2α). Blood and follicular fluid were collected 48-50 h post-administration of PGF2α. (follicular phase of the estrous cycle). Concentrations of IGF-I were measured by RJA after acid-ethanol treatment of serum or follicular fluid. Twin-producing cows had a greater (p < 0.05) number of large (>4 mm) follicles and 47% greater (p < 0.05) concentrations of IGF-I in peripheral blood than control cows. Cattle selected for high twinning frequency also had greater (p < 0.05) concentrations of IGF-I (±SE) in the two largest follicles than control (unselected) cows (327 ± 28 vs. 243 ± 29 ng/ml). IGF-I concentrations in pooled small (1-3.9 mm) follicles were less (p < 0.05) than in large follicles but did not differ between control and twin-producing cattle. In addition, the percentage of IGF-I concentrations measured in follicular fluid to that of serum was lower (P < 0.05) in small follicles than in large follicles, and was greater (p <0.05) in large follicles of control (93.2 ± 5.3%) than twin-producing (76.2 ± 4.4%) cattle. Moreover, concentrations of IGF-I in serum and follicular fluid were correlated positively (r = 0.69, p < 0.01). Concentrations of estradiol and progesterone in follicular fluid of small or large follicles did not differ between control and twin-producing cattle. Collectively, the present study provides evidence suggesting that natural twinning in cattle is associated with increased concentrations of IGF-I in both blood and follicular fluid. These data are consistent with the hypothesis that IGF-I plays a role in the regulation of folliculogenesis and is a mediator of a genetic component of multiple ovulations in cattle.