The present study was performed to determine the respective involvement of the cellular and paracellular routes in ileal Ca²⁺ transport. Two groups of rats were either fed a normal Ca²⁺ diet (1.0%) or a Ca²⁺-deficient diet (0.02%) for 14 days. Ileal Ca²⁺ absorption was determined using both an in situ method of continuous luminal perfusion and an in vitro method (Ussing chamber model). The low-Ca²⁺ diet stimulated net Ca²⁺ flux in the ileum twofold, associated with a twofold increase of the mucosal-to-serosal Ca²⁺ flux in both models. This effect was observed in the absence of concomitant changes in Na⁺ or water flux in the in situ model or mannitol flux in the in vitro model, excluding the participation of the paracellular pathway in Ca²⁺ transport. Thus only cellular Ca²⁺ flux was stimulated. These data suggest that the ileum plays a major role in the adaptation to low dietary Ca²⁺. Whereas under physiological conditions with usual Ca²⁺ intakes the transcellular pathway of Ca²⁺ transport is negligible, it becomes of major importance in the case of Ca²⁺ deficiency, at least under the present conditions of severe Ca²⁺ deprivation.