The role of estrogen in the regulation of calcium balance is still poorly understood. A calcium balance study was performed to examine the effects of estrogen status in relation to fecal calcium loss as a component of bone loss after oophorectomy (OOX) in the mature rat. The components of the classic calcium balance were compared with calcium balance estimates obtained from whole body bone density. Six month or older Sprague Dawley rats were allocated to either a sham-operated or OOX group and fed a 0.1% calcium diet. The bone mineral density (BMD) and bone mineral content (BMC) were measured at baseline, 6 weeks, and 9 weeks. A calcium balance was done for 6 days before and 6 weeks post OOX. The fall in BMD from baseline to 9 weeks in the OOX group was significantly greater than in the sham-operated group. The calcium balance was more negative at baseline than at 6 weeks in both groups of animals because they had not adapted to the low calcium diet. However, the increase in calcium balance was significantly less in the OOX animals than in the sham-operated animals. The greater the rise in calcium balance from the baseline to the 6 weeks balance the less the fall in the calcium content of the whole body (Spearman correlation: r = 0.604 P = 0.008). The fall in fecal calcium, but not urine calcium or calcium consumed, was negatively correlated with the change in whole body BMC (Spearman correlation: fecal calcium r = -0.763 P = 0.001). Thus, the primary effect of estrogen deficiency on calcium balance in the mature rat appears to be calcium flux in the bowel, rather than renal calcium handling.