High physiological prolactin (PRL) stimulated intestinal calcium absorption and renal calcium uptake in mammals. Previous histomorphometric study revealed a significant increase in bone turnover in the trabecular part of the PRL-exposed long (cortical) bone; however, whole-bone densitometric analysis was unable to demonstrate such effect. We therefore studied differential changes in bone mineral density (BMD) and contents (BMC) of the femoral diaphysis and metaphysis in adult female rats exposed to high PRL induced by anterior pituitary (AP) transplantation. The estrogen-dependent effects of PRL on the femur were also investigated. We found that chronic exposure to PRL had no effect on BMD or BMC of the femoral diaphysis, which represented the cortical part of the long bone. It is interesting that 7 weeks after an AP transplantation, BMD and BMC of the femoral metaphysis were significantly decreased by 8% and 14%, respectively. Ovariectomy (Ovx) for 2, 5, and 7 weeks also decreased BMD and BMC in the femoral metaphysis, but not in the diaphysis. However, the AP transplantation plus Ovx (AP+Ovx) produced no additive effects. Nevertheless, 2.5 μg/kg 17β-estradiol (E2) supplementation abolished the osteopenic effects of both Ovx and AP+Ovx on the femur. As for the L5–6 vertebrae, BMD and BMC were not affected by PRL exposure, but were significantly decreased by Ovx and AP+Ovx, and such decreases were completely prevented by E2 supplementation. It could be concluded that high physiological PRL induced a significant osteopenia in the trabecular part, i.e., the metaphysis, of the femora of adult female rats in an estrogen-dependent manner. Since PRL had no detectable effect on the vertebrae, the effects of PRL on bone appeared to be site-specific.