Absorption-enhancing properties of the long-chain acylcarnitines (C12-C18) were examined in vitro utilizing a modified Sweetana-Grass diffusion cell system. Transepithelial electrical resistance (TEER) and drug permeability (P) of rat colonic mucosa were used as parameters to determine the potency, selectivity and reversibility of acylcarnitine-enhancing effects. All long-chain acylcarnitines induced a rapid, concentration-dependent decrease in mucosal TEER. The minimum or threshold concentration (Ct) which produced a rapid decrease in mucosal TEER was determined for each acylcarnitine. Tissues treated with either palmitoyl or lauroyl carnitine at their Ct showed significantly better recovery of TEER after removal of the enhancers from the diffusion cells as compared to the other long-chain acylcarnitines. In addition, palmitoyl and lauroyl carnitine treatment significantly enhanced the mucosal permeability to small, hydrophilic markers without adversely altering tissue morphology. Following enhancer replacement with buffer alone and the subsequent recovery of TEER, mucosal permeability was observed to return to near control levels. At higher concentrations of palmitoyl and lauroyl (5 x Ct), P increased nearly 2-fold over that observed at the Ct; however, epithelial barrier morphology was compromised. This study shows that colonic mucosal tissue mounted in modified Sweetana-Grass diffusion cells may be a useful model for examining the mechanism(s) of absorption enhancer activity. Moreover, the results present evidence that the long-chain acylcarnitines may enhance drug absorption via two different concentration-dependent pathways.