Increasing evidence suggests that hyperinsulinemia plays an important role in the pathogenesis of polycystic ovary syndrome (PCOS). However, the timing for the onset of hyperinsulinemia is not clear. The objective of this study was to examine the effect of peripubertal hyperinsulinemia on the maturing female reproductive axis.Hyperinsulinemia was induced in 28-day-old peripubertal female rats by infusing insulin (0.04 IU/d) via subcutaneously implanted Alzet minipumps (Model #2004; Durect Corp, Cupertino, CA; constant flow rate 0.25 muL/h) for 4 weeks. Control animals were administered normal saline. Estrus cyclicity was monitored regularly. Upon termination of the experimental period, the animals were killed, trunk blood and pituitaries were collected for hormone assays, and ovaries were collected for histological and immunocytochemical studies.In contrast to the control animals, hyperinsulinemic animals had (1) erratic estrus cycles, with prolonged (2 to 3 days) metestrus-diestrus or diestrus-proestrus stages; (2) significantly (P <.05) decreased levels of serum progesterone, and significantly (P <.05) increased levels of serum testosterone and dehydroepiandrostene sulfate; (3) prematurely luteinized ovarian follicles with prominent thecal and interfollicular stromal proliferation; and (4) markedly reduced expression of growth differentiation factor-9 (GDF-9) and activin receptors (ActR) I and IB in the ovaries.Peripubertal hyperinsulinemia in rats causes hormonal and ovarian changes similar to those in women with PCOS. Based on these novel findings, we speculate that peripubertal hyperinsulinemia may be a risk factor for the development of PCOS later in life.