Since hyperprolactinemia was found to induce osteopenia in the metaphysis of long bone in non-mated female rats, pregnant and lactating rats with sustainedly high plasma prolactin (PRL) levels might also exhibit some changes in their long bones. We performed a longitudinal study in pregnant, lactating and post-weaning rats, using dual-energy X-ray absorptiometry to demonstrate site-specific changes (i.e., metaphysis vs. diaphysis) in femoral bone mineral density (BMD) and content (BMC). The results showed that femoral metaphyseal BMD and BMC were higher when compared to their age-matched controls during pregnancy, before decreasing in late lactation and post-weaning. On the other hand, femoral diaphyseal BMC increased during pregnancy, early lactating and mid-lactating periods without change during late lactation and post-weaning. After 7 days of bromocriptine administration which inhibited endogenous PRL secretion, the lactation-induced increases in BMC during early and mid-lactating periods were abolished. Moreover, a decrease in metaphyseal BMD during late lactation was restored to the control levels by bromocriptine. However, bromocriptine did not antagonize the pregnancy-induced increases in BMD and BMC. It could be concluded that the effect of PRL on bone was variable during the reproductive periods. While having no effect on femoral BMD and BMC during pregnancy, PRL was responsible for bone gain in early and mid-lactating periods, but induced bone loss during late lactating period.