PURPOSE: Lactation often affects calcium metabolism and induces bone loss. Calcium supplementation and a high calcium diet are recommended to prevent bone loss, especially during inadequate calcium intake. Our study aimed at determining bone loss in breastfeeding mothers, and if it occurred, whether it was site specific and there were correlations between serum bone turnover markers. MATERIALS AND METHODS: Since the 6-month exclusive breastfeeding is usually recommended in several countries, our study examined bone mineral density (BMD) in early (1-2 month), mid (3-4 month)-, and late (5-6 month) lactation compared with nonpregnant, nonlactating control women. Site-specific bone loss was monitored in lumbar vertebrae and femora. Bone turnover markers, that is, C-terminal telopeptide of type 1 collagen and N-terminal propeptide of type 1 collagen (P1NP), were determined by electrochemiluminescence immunoassays. RESULTS: The onset of bone loss in exclusive breastfeeding mothers was site specific, for example, in the lumbar bone at mid-lactation and in the femoral bone in late lactation. Serum ionized calcium levels in late lactation were lower than the normal levels. In addition, a correlation was found between bone turnover marker, P1NP, and femoral BMD. CONCLUSIONS: The onset of bone loss in exclusive breastfeeding mothers was site specific, and the lumbar bone was a vulnerable and perhaps better representative site for bone loss detection. It was suggested that the optimal starting time for calcium supplementation should be before the mid-lactation when the bone loss was observed. In addition, the biochemical marker that best predicted the onset of bone loss in lactating women was P1NP.