The strategic localization of peptide transporter 2 (PEPT2), a proton-coupled oligopeptide transporter, to the apical membrane of epithelial cells in the kidney and choroid plexus suggests that it plays an important role in the disposition of peptides/mimetics in the body. Therefore, the in vivo significance of PEPT2 was investigated in wild-type and PEPT2 null mice following an i.v. bolus dose (0.05 micromol/g body weight) of [14C]glycylsarcosine (GlySar). In PEPT2 null mice, the clearance (total and renal) of GlySar was markedly increased (2-fold), resulting in concomitantly lower systemic concentrations. In addition, renal reabsorption was almost abolished, and GlySar was eliminated by glomerular filtration. Of the 46% of GlySar reabsorbed in wild-type mice, PEPT2 accounted for 86% and PEPT1 accounted for 14% of reabsorbed substrate. Analysis of GlySar uptake in kidney sections revealed that PEPT2 was primarily localized in the outer medullary region. Wild-type mice also had greater choroid plexus concentrations of GlySar and a 5-fold greater choroid plexus/cerebrospinal fluid (CSF) ratio as compared with null mice at 60 min. Null mice exhibited a greater CSF/blood ratio at 60 min (0.9 versus 0.2) and area under the curve (AUC)(CSF)/AUC(blood) ratio over 60 min (0.45 versus 0.12), indicating that PEPT2 significantly reduces the exposure of GlySar in CSF. Our in vivo results demonstrate that PEPT2 is the predominant peptide transporter in kidney and that it acts as an efflux transporter in choroid plexus. Thus, PEPT2 may have profound effects on the sensitivity and/or toxicity of peptides and peptide-like drugs.