The limited oral bioavailability of the potent and selective group II metabotropic glutamate (mGlu) 2/3 receptor agonist, (1S,2S,5R,6S)-2- aminobicyclo[3.1.0]hexane-2,6-dicarboxylate (LY354740), was shown to be improved by its peptidyl prodrug, (1S,2S,5R,6S)-2-[(2′S)-(2′-amino) propionyl]aminobicyclo[3.1.0]hexane-2,6-dicarboxylate (LY544344). The purpose of this study was to elucidate the mechanisms of intestinal absorption of LY354740 and its prodrug LY544344. Transepithelial transport and accumulation studies were performed in Caco-2 cell monolayers; the involvement of the peptide transporter 1 (PEPT1) transporter was also examined. In absorptive transport studies, the peptidyl prodrug LY544344 partially hydrolyzed to release LY354740 intracellularly, and both compounds appeared in the basolateral compartment. The absorptive transport rate of LY544344, basolateral appearance rate of LY354740, and their cellular accumulation after incubation with LY544344 were concentrationdependent. PEPT1 inhibition reduced transepithelial transport and cellular accumulation of LY544344 to 22 and 1.1 % of control, respectively. LY354740 showed concentration-independent absorptive transport with negligible cellular accumulation. Efflux and transstimulation studies revealed predominantly apical efflux and the existence of specific transporters for LY544344 and intracellularly released LY354740 on the apical and basolateral membranes. LY544344 efflux was also trans-stimulated at the apical side by glycyl-glutamate but not by glycyl-sarcosine. Transport of neither compound was affected by P-glycoprotein-mediated efflux, as shown in transport and uptake inhibition studies in Madin-Darby canine kidney multidrug resistance 1-transfected cell line and inverted membrane vesicles. In conclusion, LY354740 is mainly transported by the paracellular pathway, whereas intestinal absorption of LY544344 is mediated by PEPT1. However, the absorptive transport of LY544344 seems to be modulated by an apical efflux transporter and a rate-limiting transport step across the basolateral membrane.