Rats were fed a purified diet containing 30% lactose and calcium absorption was measured in duodenal loops in situ following instillation of 1.25 or 10 mM CaCl2 solutions. Lactose feeding caused absorption to be depressed from 88 to 69% (1.25 mM Ca solution) and from 71 to 43% (10 mM Ca solution). The effect of lactose feeding was more pronounced in 5-month old rats than in 2-month old rats. In the lactose-fed rats, calcium-binding protein (CaBP), measured by a competitive binding assay following partial purification, was depressed on the average from 24 to 10 nmoles Ca bound per mg protein. The effect of the lactose ingestion can be likened to the effect expected from continued high calcium intake, i.e., a decrease in the efficiency of calcium absorption and a decrease in CaBP.