Abstractl‐Kyotorphin (l‐KTP), an endogenous analgesic neuropeptide, is a substrate for aminopeptidases and a proton‐coupled oligopeptide transporter, PEPT2. This study examined the CSF efflux, antinociceptive response, and hydrolysis kinetics in brain ofl‐KTP and its synthetic diastereomerd‐kyotorphin (d‐KTP) in wild‐type andPept2null mice. CSF clearance ofl‐KTP was slower inPept2null mice than in wild‐type animals, and this difference was reflected in greaterl‐KTP‐induced analgesia inPept2null mice. Moreover, dose‐response analyses showed that the ED50 ofl‐KTP inPept2‐deficient animals was one‐fifth of the value observed inPept2‐competent animals (4 vs. 21 nmol for null vs. wild‐type mice, respectively). In contrast, the ED50 ofd‐KTP was very similar between the two genotypes (9–10 nmol). Likewise, there was little difference between genotypes in slope factor or baseline effects ofl‐KTP andd‐KTP. The enhanced antinociceptive response tol‐KTP inPept2null mice could not be explained by differences in neuropeptide degradation as Vmax and Km values did not differ between genotypes. Our results demonstrate that PEPT2 can significantly impact the analgesic response to an endogenous neuropeptide by altering CSF (and presumably brain interstitial fluid) concentrations and that it may influence the disposition and response to exogenous peptide/mimetic substrates.