AbstractN‐arachidonyl‐glycine is one of a series of N‐arachidonyl‐amino acids that are derived from arachidonic acid. N‐arachidonyl‐glycine is produced in a wide range of tissues with greatest abundance in the spinal cord. Here we report that N‐arachidonyl‐glycine is a reversible and non‐competitive inhibitor of glycine transport by GLYT2a, but has little effect on glycine transport by GLYT1b or γ‐amino butyric acid transport by GAT1. It has previously been reported that the activity of GLYT2a is down‐regulated by protein kinase C and therefore we investigated whether the actions of N‐arachidonyl‐glycine on GLYT2a are mediated by second messenger systems that lead to the activation of protein kinase C. However, the protein kinase C inhibitor, staurosporine, had no effect on the actions of N‐arachidonyl‐glycine on GLYT2a. Thus, the actions of N‐arachidonyl‐glycine are likely to be mediated by a direct interaction with the transporter. We have further defined the pharmacophore by investigating the actions of other N‐arachidonyl amino acids as well as the closely related compounds arachidonic acid, anandamide and R1‐methanandamide. Arachidonic acid, anandamide and R1‐methanandamide have no effect on glycine transport, but N‐arachidonyl‐l‐alanine has similar efficacy at GLYT2a to N‐arachidonyl‐glycine, and N‐arachidonyl‐γ‐amino butyric acid is less efficacious. These observations define a novel recognition site for the N‐arachidonyl amino acids.