Lactating rats fed a vitamin D-containing diet maintain elevated serum levels of 1,25-dihydroxycholecalciferol [1,25-(OH)2D3] and increased intestinal net calcium absorption (as a percentage of the intake). Rts deprived of vitamin D for only 2 weeks during lactation lack elevated serum levels of 1,25-(OH)2D3 and develop marked hypocalcemia. We have now determined net absorption of Ca for a 3-day period at the end of lactation in rats suckling a second litter of 10-day-old pups. The rats were fed diets containing either no vitamin D (--D) or 5 IU vitamin D3/g diet (+D) from the sixth day of pregnancy. Net Ca absorption was at least 2-fold higher in +D-fed lactating rats than in nonlactating controls. Vitamin D-deprived lactating rats had values approximately the same as the +D-fed lactating rats, even though the former were severely hypocalcemia and lacked elevated levels of 1,25-(OH)2D3 in plasma and intestinal mucosa. The high efficiency of intestinal Ca absorption in --D lactating rats was confirmed in vitro with everted duodenal sacs which developed essentially the same high Ca concentration ratios across the intestinal wall as did sacs from +D rats. Parathyroidectomy did not significantly affect the Ca concentration ratio of sacs obtained from --D lactating rats 2 days after the operation. We conclude that during lactation, 1,25-(OH)2D3 is primarily required for calcium homeostasis, and enhanced calcium absorption does not require elevated serum and intestinal levels of 1,25-(OH)2D3.