Pubertal mammary development in the rat is largely dependent upon GH and estrogen. We recently showed that insulin-like growth factor-I (IGF-I) can substitute for GH in inducing mammary development in male rats, suggesting that IGF-I mediates GH action. The present study investigated whether IGF-I, like GH, required estradiol (E2) to act or whether IGF-I could substitute for both GH and E2. The effects of IGF-I were tested in the presence and absence of E2. Elvax pellets containing IGF-I or des(1-3) IGF-I were implanted into right lumbar mammary glands of sexually immature, hypophysectomized, oophorectomized female rats, with control BSA-containing pellets in the contralateral glands. After 5 days, both lumbar mammary glands were removed and examined in whole mounts for mammary development by counting terminal end buds and alveolar structures. E2, administered in SILASTIC brand capsules, had no independent effect on mammary development. In the absence of E2, des(1-3) IGF-I had a small, but significant, independent effect on mammary development; native IGF-I was ineffective. The addition of E2 significantly enhanced the effects of IGF-I and des(1-3) IGF-I on mammary development, similar to that noted when E2 was given along with GH. We also studied the effects of E2 and/or hGH on mammary gland IGF-I messenger RNA (mRNA) in hypophysectomized castrated male animals. E2 alone did not increase mammary gland IGF-I mRNA concentrations, but E2 enhanced the effect of hGH on IGF-I mRNA by 4- to 6-fold. These studies indicate that IGF-I can have a small independent effect on mammary development, but like GH, E2 is required for a full effect. They also indicate that E2 is capable of synergizing with GH in the production or expression of IGF-I mRNA, and that the action of E2 on mammary development may take place at multiple sites. If locally produced IGF-I does indeed mediate the action of GH in mammary development, then although E2 is capable of enhancing the effect of GH on IGF-I mRNA, its major effect in mammary development occurs after IGF-I is produced.