γ-Hydroxybutyric acid (GHB) is a recreational drug， a clinically prescribed drug in narcolepsy and alcohol dependence， and an endogenous substance that binds to both high- and low-affinity sites in the brain. For studying the molecular mechanisms and the biologic role of the GHB high-affinity binding sites， ligands with high and specific affinity are essential. The conformationally restricted GHB analog HOCPCA (3-hydroxycyclopent-1-enecarboxylic acid) is one such compound. The objective of this study was to investigate the transport of HOCPCA across the blood-brain barrier in vitro and in vivo and to investigate the hypothesis that HOCPCA， like GHB， is a substrate for the monocarboxylate transporters (MCTs). For in vitro uptake studies， MCT1， -2， and -4 were recombinantly expressed in Xenopus laevis oocytes， and the previously reported radioligand [ H]HOCPCA was used as substrate. HOCPCA inhibited the uptake of the endogenous MCT substrate l-[ C]lactate， and [ H]HOCPCA was shown to act as substrate for MCT1 and 2 (Km values in the low- to mid-millimolar range). Introducing single-point amino acid mutations into positions essential for MCT function supported that HOCPCA binds to the endogenous substrate pocket of MCTs. MCT1-mediated brain entry of HOCPCA (10 mg/kg s.c.) was further confirmed in vivo in mice by coadministration of increasing doses of the MCT inhibitor AR-C141990 [(R)-5-(3-hydroxypyrrolidine-1-carbonyl)-1-isobutyl-3-methyl-6-(quinolin-4-ylmethyl)thieno[2，3-d]pyrimidine-2，4(1H，3H)-dione]， which inhibited brain penetration of HOCPCA in a dose-dependent manner (ID50 = 4.6 mg/kg). Overall， our study provides evidence that MCT1 is an important brain entry site for HOCPCA and qualifies for future in vivo studies with HOCPCA.