In the forebrain, synaptic glycine concentrations are regulated through the glycine transporter GlyT1. Because glycine is a coagonist of the N-methyl-D-aspartate (NMDA) receptor (NMDAR), which has been implicated in schizophrenia, inhibition of GlyT1 is thought to provide an option for the treatment of schizophrenia. In support of this hypothesis, GlyT1 inhibitors facilitate in vivo NMDAR function and demonstrate antipsychotic-like effects in animal models. Among the specific GlyT1 inhibitors, substituted N-methyl-glycine (sarcosine) derivatives (e.g., (R)-N[3-(4'fluorophenyl)-3-(4'phenyl-phenoxy)propyl]-sarcosine [NFPS], (R)-N[3-phenyl-3-(4'-(4-toluoyl)phenoxy)-propyl]sarcosine [(R)-NPTS], and (R,S)-(+/-)N-methyl-N-[(4-trifluoromethyl)phenoxy]-3-phenyl-propylglycine [Org24589]), and non-sarcosine-containing inhibitors, such as 2-chloro-N-[(S)-phenyl[(2S)-piperidin-2-yl] methyl]-3-trifluoromethyl benzamide, monohydrochloride (SSR504734), have been described. In the present study, we analyzed the mode of interaction of these compounds with GlyT1 by using electrophysiological measurements in Xenopus laevis oocytes, and with two binding assays, using [(3)H](R)-NPTS or 2-chloro-N-[(S)-phenyl[(2S)-N-methylpiperidin-2-yl]-methyl]-3-trifluoromethyl benzamide monohydrochloride ([(3)H]N-methyl-SSR504734) as radioligands. Inhibition of electrogenic glycine transport by sarcosine-based compounds was apparently irreversible and independent of glycine concentration. The latter indicates a noncompetitive mode of action. In contrast, both SSR504734 and N-methyl-SSR504734 exhibited reversible and competitive inhibition of glycine transport. In GlyT1-expressing membranes, the binding of the novel radioligand [(3)H]N-methyl-SSR504734 to a single site on GlyT1 was competitively displaced by glycine and SSR504734 but noncompetitively by sarcosine-based compounds. Inversely, [(3)H](R)-NPTS binding was competitively inhibited by sarcosine-based compounds, whereas glycine, SSR504734, and N-methyl-SSR504734 noncompetitively decreased maximal binding. Our data indicate that besides exerting an apparently irreversible or reversible inhibition, GlyT1 inhibitors differ by exhibiting either a noncompetitive or competitive mode of inhibition. The divergent modes of inhibition may significantly affect the efficacy and tolerability of these drugs.