The overall objective of this study was to determine the mechanisms of acylcarnitine-mediated enhancement of calcium transport across Caco-2 cells. The different mechanisms of enhancement postulated are (a) loosening of tight junctions, thereby promoting paracellular transport; (b) opening of calcium channels, thus increasing calcium entry; and (c) stimulation of the basolateral Ca-ATPase pump, thereby aiding calcium extrusion. Although the existence of calcium channels and the reversal of verapamil-mediated inhibition of calcium uptake by acylcarnitines were demonstrated for the first time in Caco-2 cells, the channels do not appear to be a major contributing factor to the enhancement of calcium transport by acylcarnitines. Calmidazolium, a potent Ca-ATPase pump inhibitor in tissues such as rat intestinal segments, failed to inhibit this pump in Caco-2 cells. Thus, the predominant mechanism of enhancement of calcium transport by acylcarnitines in the Caco-2 model appears to be via promotion of paracellular transport.