Anandamide (AEA) and delta9-tetrahydrocannabinol (THC) are endogenous and exogenous ligands, respectively, for cannabinoid receptors. Whereas most of the pharmacological actions of cannabinoids are mediated by CB1 receptors, there is also evidence that these compounds can produce effects that are not mediated by the activation of identified cannabinoid receptors. Here, we report that THC and AEA, in a CB1 receptor-independent manner, cause a significant potentiation of the amplitudes of glycine-activated currents (I(Gly)) in acutely isolated neurons from rat ventral tegmental area (VTA) and in Xenopus laevis oocytes expressing human homomeric (alpha1) and heteromeric (alpha1beta1) subunits of glycine receptors (GlyRs). The potentiation of I(Gly) by THC and AEA is concentration-dependent, with respective EC50 values of 86 +/- 9 and 319 +/- 31 nM for alpha1 homomeric receptors, 73 +/- 8 and 318 +/- 24 nM for alpha1beta1 heteromeric receptors, and 115 +/- 13 and 230 +/- 29 nM for native GlyRs in VTA neurons. The effects of THC and AEA are selective for I(Gly), because GABA-activated current in VTA neurons or in X. laevis oocytes expressing alpha2beta3gamma2 GABA(A) receptor subunits were unaffected by these compounds. The maximal potentiation by THC and AEA was observed at the lowest concentration of glycine; with increasing concentrations of glycine, the potentiation significantly decreased. The site for THC and AEA seems to be distinct from that of the alcohol and volatile anesthetics. The results indicate that THC and AEA, in pharmacologically relevant concentrations, directly potentiate the function of GlyRs through an allosteric mechanism.