Concentration and voltage dependence of unidirectional 45Ca transport measurements indicated that approximately 60-70% of the mucosa-to-serosa calcium flux measured across the short-circuited rat duodenum, jejunum and ileum is paracellular, with only 30-40% of the mucosa-to-serosa calcium transport cellular. The calcium flux from serosa to mucosa was purely paracellular in all segments. Duodenal calcium serosal-to-mucosal flux was of the same order of magnitude as the mucosal-to-serosal paracellular movement. However, the serosal-to-mucosal flux of jejunum and ileum was twice as high. Therefore, net calcium absorption occurs only in the duodenum, whereas calcium is secreted in the jejunum and ileum by a passive paracellular route, presumably involving an anomalous solvent drag effect. Administration of 1,25-dihydroxycholecalciferol (1,25-(OH)2D3) led to an increase in the transcellular mucosa-to-serosa flux in the duodenum only. It also led to a stimulation of paracellular calcium flux in both directions in all three intestinal segments, with no change in net paracellular calcium absorption. Thus, the only vitamin D-related increase in calcium absorption was due to the increase in duodenal transcellular absorption. The mechanism by which 1,25-(OH)2D3 increased paracellular flux is not known but may have resulted from an osmotic effect on the intercellular spaces.