Previous studies of dietary protein intake and calcium balance demonstrate decreased fecal calcium excretion with high-protein diets. To determine whether a duodenal or ileal calcium transport response could account for these findings, we examined calcium transport by these two segments directly by in situ perfusion. Weight-matched growing rats were pair-fed isocaloric diets for 6-8 days containing 89% protein (casein), 39% and 0% protein. Sucrose was substituted for the decreased casein in the 39% and 0% protein diets. Each diet also contained 5% fat, a mineral mix, and vitamins. Calcium content of all diets was 0.5%. Body weight was maintained by the 39% protein group; weight loss was 5% for the 89% protein group and 18% for the 0% protein group. Small-intestinal weight as a percentage of body weight was the same for the three groups. For both the duodenum and the ileum, mucosal dry weight per centimeter was greater in the 39% protein group than either the 89% protein or 0% protein group. Calcium absorption per gram dry weight of mucosa (absorptive specific activity) was the same for all diet groups, but because of the greater mucosal weight per unit length in the 39% protein group, absorption per centimeter was increased in this group. In conclusion, although we found no direct relation between dietary protein and mucosal absorptive specific activity for calcium, calcium absorption was determined by the amount of mucosa which was dependent on protein intake and reflected the nutritional status of the body.