We have developed a simple test of fractional calcium absorption in rats using oral 45Ca. The tracer was administered via a gastric tube, each animal receiving 0.15 microgram 45Ca with calcium glubionate as a carrier. Plasma radioactivity was determined at 30, 60, 90 and 120 min and expressed as percent dose in extracellular fluid. The fractional absorption of 45Ca was also expressed as 'area under the curve' (F45Ca) calculated by Simpson's approximation for each rat. The fractional absorption of calcium was determined in fasting and nonfasting fed animal subsets, each subset including control and rats treated with 1,25(OH)2D3. After 1,25(OH)2D3 administration, a significant increase in plasma calcium was seen in both fasting and fed animals. The fractional absorption of calcium was significantly higher in 1,25(OH)2D3-treated fasting as compared with control rats. In these animals, a positive correlation was found between F45Ca and plasma calcium. In contrast, in fed rats, the determined fractional absorption of calcium was similar in control and after 1,25(OH)2D3 administration. These results point to the use of orally administered 45Ca as a technically easy method for calcium absorption which can be used in most laboratories. In fasting animals it is sensitive enough for the evaluation of the effect of promoters of calcium absorption. The different findings in fed and fasting states may be related to the transmural processes which regulate the intestinal absorption of calcium.