Glycine receptors (GlyRs) mediate inhibitory neurotransmission in spinal cord motor and pain sensory neurons. Recent studies demonstrated apparently contradictory (potentiating versus inhibitory) effects of the endocannabinoid anandamide on these receptors. The present study characterised the effects of cannabinoid agonists on α1, α1β, α2 and α3 GlyRs recombinantly expressed in HEK293 cells with the aims of reconciling effects of cannabinoids on these receptor subtypes and to establish the potential of different GlyR isoforms as novel physiological or analgesic targets for cannabinoids. The compounds investigated were anandamide, HU-210, HU-308, WIN55,212-2 and the endogenous non-cannabinoid, N-arachidonyl-glycine. The latter compound was chosen due to the structural similarity with anandamide and known analgesic actions in the spinal cord. Recombinant α1 and α1β GlyRs were potentiated by anandamide and HU-210 at submicromolar concentrations, whereas WIN55,212-2 had no effect and HU-308 produced only weak inhibition. By contrast, N-arachidonyl-glycine exerted complex effects including both potentiation and inhibition. Anandamide had no effect at α2 or α3 GlyRs although the other cannabinoids produced potent inhibition. On α2 GlyRs, the inhibitory potency sequence was HU-210=WIN55,212-2>HU-308>N-arachidonyl-glycine but on α3 GlyRs it was HU-210=WIN55212=HU-308>N-arachidonyl-glycine. These results suggest that α1, α2 and α3 containing GlyRs exhibit distinct pharmacological profiles for cannabinoids. We conclude that cannabinoid agonists may be useful as pharmacological tools for selectively inhibiting α2 and α3 GlyRs. Our results also establish GlyRs as potential novel targets for endogenous and exogenous cannabinoids.