Calcium and magnesium transport at different luminal concentrations (1.25, 2.5, 5 and 10 mmol/l) and under the influence of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] were studied in vivo at the perfused colon of the rat. Net absorption of calcium and of magnesium were saturated with increasing concentrations of the perfusate. Net absorption of one ion was not affected by an increasing concentration of the other ion. 1,25(OH)2D3 (100 ng/day given subcutaneously for 4 days) stimulated net absorption of calcium and of magnesium. Presence or absence of calcium or magnesium, respectively, had no effect on the absorption of the other ion under 1,25(OH)2D3 stimulation. The results demonstrate that the colon of the rat is capable of absorbing calcium as well as magnesium. These observations suggest that calcium and magnesium in the rat colon are absorbed by two separate active transport mechanisms, which both are sensitive to 1,25(OH)2D3.