The effects of varying dietary calcium and phosphorus content, vitamin D deficiency, oophorectomy, adrenalectomy, and simultaneous pregnancy on bone mineral loss during lactation were examined in rats. Unless otherwise stated, the diet contained 0.47% calcium and 0.3% phosphorus and the rats were given 26 nmol of vitamin D3. Femur ash weights were determined after 21 days of lactation and on age-matched nonlactating rats. Decreasing dietary calcium to 0.02% caused an increased loss of bone mineral, whereas increasing dietary calcium to 1.4% increased plasma calcium levels to 12 mg/100 ml but did not diminish the bone mineral loss observed during lactation. Varying dietary phosphorus did not have a major effect on bone mineral loss during lactation. In vitamin D-deficient rats, bone mineral loss during lactation was independent of dietary calcium levels and slightly greater than the loss observed in vitamin D-replete rats fed the normal calcium diet. Oophorectomy and adrenalectomy did not produce changes in femur ash weights of nonlactating rats or reduce bone mineral loss during lactation. Rats mated during their postpartum estrus and thus simultaneously pregnant and lactating, lost the same amount of bone mineral as caused by lactation alone.