Acylcarnitines with chain lengths of 2 to 18 carbon atoms were tested for their effects on rat intestinal brush border membrane order (S) by fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH). These results were compared to the previously reported effectiveness of the acylcarnitines as absorption enhancers of the poorly absorbed antibiotic cefoxitin. Acylcarnitines with fatty acids less than 12 carbon units in length were ineffective in increasing drug absorption and perturbing brush border membrane order. Long-chain acylcarnitines (12-18 carbons) significantly increased the bioavailability of cefoxitin and decreased the lipid order of brush border membranes. The results suggest that, in order to promote drug absorption, the acylcarnitines must surpass a critical chain length (10 carbon units) to partition effectively into the membrane and, in addition, must perturb the lipid order beyond a threshold value (15-20%). Membrane perturbing capacity may serve as an indicator of the absorption enhancing potential of other aliphatic-type compounds.