While prolactin-induced increase in trabecular calcium deposition was absent after ovariectomy, its effects on cortical bones were controversial. This investigation aimed to study the effect of prolactin induced by either pituitary (AP) transplantation or prolactin injection on cortical bones in ovariectomized rats. Since the presence of prolactin receptors (PRLR) in different bones of adult rats has not been reported, we first determined mRNA expression of PRLRs at the cortical and trabecular sites by using the RT-PCR. Our results showed the PRLR mRNA expression with predominant long-form at all sites. However, high prolactin induced by AP transplantation in normal rats did not have any effect on the femoral BMD. By using ⁴⁵Ca study, prolactin did not alter bone formation, bone resorption and calcium content in tibia and femur of adult ovariectomized rats. AP transplantation also had no effect on the cortical calcium content in adult ovariectomized rats. Because previous work showed that the effects of prolactin were age-dependent and could be modulated by high-calcium diet, interactions between prolactin and these two parameters were investigated. The results demonstrated that high-calcium diet increased the tibial calcium content in young AP-grafted ovariectomized rats, but decreased the tibial calcium content in adult rats. As for the vertebrae, the calcium contents in both young and adult rats were not changed by high-calcium diet. Our results thus indicated that the adult cortical bones were potentially targets of prolactin. The effects of prolactin on cortical bones were age-dependent and were observed only under the modulation of high-calcium diet.