Background and PurposeAlthough cannabinoid CB2 receptor ligands have been widely characterized in recombinant systems in vitro, little pharmacological characterization has been performed in tissues natively expressing CB2 receptors. The aim of this study was to compare the pharmacology of CB2 receptor ligands in tissue natively expressing CB2 receptors (human, rat and mouse spleen) and hCB2‐transfected CHO cells.Experimental ApproachWe tested the ability of well‐known cannabinoid CB2 receptor ligands to stimulate or inhibit [35S]GTPγS binding to mouse, rat and human spleen membranes and to hCB2‐transfected CHO cell membranes. cAMP assays were also performed in hCB2‐CHO cells.Key ResultsThe data presented demonstrate that: (i) CP 55,940, WIN 55,212‐2 and JWH 133 behave as CB2 receptor full agonists both in spleen and hCB2‐CHO cells, in both [35S]GTPγS and cAMP assays; (ii) JWH 015 behaves as a low‐efficacy agonist in spleen as well as in hCB2‐CHO cells when tested in the [35S]GTPγS assay, while it displays full agonism when tested in the cAMP assay using hCB2‐CHO cells; (iii) (R)‐AM 1241 and GW 405833 behave as agonists in the [35S]GTPγS assay using spleen, instead it behaves as a low‐efficacy inverse agonist in hCB2‐CHO cells; and (iv) SR 144528, AM 630 and JTE 907 behave as CB2 receptor inverse agonists in all the tissues.Conclusion and ImplicationsOur results demonstrate that CB2 receptor ligands can display differential pharmacology when assays are conducted in tissues that natively express CB2 receptors and imply that conclusions from recombinant CB2 receptors should be treated with caution.