Concentrations of extracellular glycine in the CNS are regulated by two Na(+)/Cl(-) -dependent glycine transporters, GlyT1 and GlyT2. Selective inhibitors of GlyT1 have been developed for the treatment of schizophrenia, whilst selective inhibitors of GlyT2 are analgesic in animal models of pain. We have assessed a series of endogenous lipids as inhibitors of GlyT1 and GlyT2. Human GlyT1 and GlyT2 were expressed in Xenopus laevis oocytes, and the inhibitory actions of a series of acylcarnitines on glycine transport were measured using electrophysiological techniques. Oleoyl-L-carnitine inhibited glycine transport by GlyT2, with an IC(50) of 340 nM, which is 15-fold more potent than the previously identified lipid inhibitor N-arachidonyl-glycine. Oleoyl-L-carnitine had a slow onset of inhibition and a slow washout. Using a series of chimeric GlyT1/2 transporters and point mutant transporters, we have identified an isoleucine residue in extracellular loop 4 of GlyT2 that conferred differences in sensitivity to oleoyl-L-carnitine between GlyT2 and GlyT1. Oleoyl-L-carnitine is a potent non-competitive inhibitor of GlyT2. Previously identified GlyT2 inhibitors show potential as analgesics and the identification of oleoyl-L-carnitine as a novel GlyT2 inhibitor may lead to new ways of treating pain.