Abstract In vitro and in situ intestinal transport of β-lactam antibiotics in the presence of two novel pharmaceutical excipients, caprylocaproyl and lauroyl macrogolglycerides (Labrasol ® and Gelucire 44/14), is described. The objective was to compare the effects of both macrogolglycerides on the intestinal transport of cephalexin, a substrate of oligopeptide transporters, and cefoperazone, a non-substrate of them. The in vitro transport studies were performed using a sheet of rat jejunum mounted in Ussing-type diffusion chambers. The in situ studies used an isolated internal loop model in the rat. Labrasol ® and Gelucire 44/14 were used as the excipients at low concentrations (0.01–0.5%, w/v). The membrane permeability of both drugs was compared by apparent permeability coefficients ( P app) determined from changes in the amount of permeation vs. time in in vitro studies and by apparent absorptive clearance ( CL app) determined from changes in the steady state drug concentration of perfusate in in situ studies. The P app value of cephalexin increased with an increase in the concentration of Labrasol ® (0.05–0.5%) compared to the value without Labrasol ®. The enhancing effect of Labrasol ® on cephalexin transport was similarly observed in in situ studies, and when 0.5% Labrasol ® was used in the presence of glycyl- l-leucine or l-alanyl- l-alanine, 60 or 46% enhancement of the active transport of cephalexin by Labrasol ® was obtained. On the other hand, Gelucire 44/14 did not affect the P app and CL app of either drug. The different effects of the excipients on cephalexin transport were thought to be due to the influences of size parameters such as a polydispersity index and particle size, and the change in the short-circuit current of jejunum by the addition of the excipient.