Additional calcium is required during lactation, and several calcium regulatory factors are involved in calcium balance. In lactating rural women who have marginal nutrition and consume a high-fiber diet, negative calcium balance may be expected. We evaluated calcium balance and its association with potential calcium regulatory factors in lactating, rural Mexican women who had marginal nutrition and consumed a high-fiber diet. This cross-sectional study included women at 1, 3, 6, and 12 mo of lactation (L1, L3, L6, and L12 groups) and women who had weaned their infants (W group). Age-matched, nonlactating women (NL group) were also included. Calcium balance and concentrations of calcium regulatory factors were determined. Correlation analysis was performed by using data from all of the lactating women. Calcium balance in the L1, L3, and L6 groups was negative and was significantly different (P < 0.05) from that in the W and NL groups. Serum parathyroid hormone (PTH) and 1,25-dihydroxyvitamin D [1,25-(OH)(2)D] concentrations were significantly higher (P < 0.05) in the W group than in the L and NL groups. Calcium balance was positively associated with serum estradiol concentrations (r = 0.58, P < 0.05) and negatively associated with serum 1,25-(OH)(2)D concentrations (r = -0.52, P < 0.05). Breast-milk calcium concentrations correlated positively with serum PTH-related peptide (PTHrP) concentrations (r = 0.51, P < 0.05) and negatively with serum estradiol concentrations (r = -0.57, P < 0.05). Negative calcium balance was observed during lactation in rural Mexican women who consumed a high-fiber diet. Furthermore, the data suggest that the hormones estradiol and PTHrP are involved in the regulation of calcium balance and of the calcium content of milk during lactation.