Using an in vitro gut-sac technique, this study examined the mechanisms of calcium (Ca) uptake along the gastrointestinal tract (GIT) of rainbow trout Oncorhynchus mykiss. Ca uptake into three different compartments (mucous-bound, mucosal epithelium and blood space) of four distinct GIT segments (stomach, anterior intestine, mid intestine and posterior intestine) was monitored after luminal exposure to 10 mM Ca saline (radiolabelled with 45Ca). Ca transport was determined to be both time-dependent and concentration-dependent. The concentration-dependent kinetics of Ca uptake was investigated using varying luminal concentrations of Ca (1, 10, 30, 60 and 100 mM). In the blood-space compartment, Ca uptake was saturable at high Ca concentrations in the mid intestine (suggesting mediated transport), while linear uptake was found in the other gut segments. In the mucous-bound and mucosal epithelium compartments, however, saturation kinetics were found for most GIT segments, also suggesting mediated transport. Manipulation of serosal saline osmotic pressure with mannitol demonstrated that Ca uptake was not greatly affected by solvent drag. Elevated mucosal cadmium (Cd) did not appear to inhibit Ca uptake into the blood space in any of the GIT sections, and Ca uptake did not appear to be sodium dependent. Maximum transport capacities for Ca and Cd were found to be comparable between the gills and gut, but affinities were much higher at the gills (up to 3000 times).