OBJECTIVE Recent studies have shown the role of human colon in the absorption of calcium, especially in the presence of severe disease or resection of the small bowel. The aim of the current study was to explore the mechanism(s) of calcium uptake by the purified human colonic apical membrane vesicles. METHODS Apical membrane vesicles were purified from mucosal scrapings of colons from organ donors, and 45Ca uptake was measured using a rapid filtration technique. RESULTS The majority of the 45Ca associated with vesicles represented uptake into closed intravesicular space, whereas the remaining 45Ca uptake represented binding to the vesicles. Ca2+ uptake was found to be dependent on time, pH, temperature, and ionic strength of the incubation medium and inhibitable by ruthenium red, La3+, and ethylene glycol-bis(beta-amino-ethyl ether)-N,N,N',N'-tetraacetic acid. Experiments of the effects of membrane potential generated by K+/valinomycin or anion substitutions on Ca2+ uptake showed that the Ca2+ uptake process was potential insensitive. Calcium uptake was unaffected by outwardly directed H+, K+, and Na+ gradients. Ca2+ uptake showed saturation kinetics with no significant differences in Michaelis constant and maximum velocity values of this transporter between proximal and distal colonic segments. CONCLUSIONS The uptake of Ca2+ by human colonic apical membranes involves predominantly a carrier-mediated transport mechanism.