AbstractG‐protein coupled receptors exist in both high and low agonist affinity conformations, with tracer levels of agonist radioligands preferentially binding to the former. The goal of the present study was to characterize the in vivo binding of the aminoalkyindole‐based, CB1 receptor agonist,R‐[125/131I]AM2233 ((2‐[125/131I]iodo‐phenyl)‐[1‐(1‐methyl‐piperidin‐2‐yl‐methyl)‐1H‐indol‐3‐yl]‐methanone), and to use this radiotracer to selectively measure the receptor occupancy by the related CB1 receptor agonist, WIN55212‐2, to the agonist‐preferring affinity state of the receptor. In mouse locomotor assays, both WIN55212‐2 and AM2233 (racemic) produced an ∼60% reduction in activity at 1 mg/kg, (i.v.) and completely inhibited activity at 3 mg/kg, confirming their agonist nature. In ex vivo autoradiography, preferential uptake ofR‐[131I]AM2233 was apparent in CB1 receptor‐rich areas, including globus pallidus, substantia nigra, striatum, cerebellum, and hippocampus. Overall brain uptake ofR‐[131I]AM2233 was 1.3% injected activity/g at 5 min in mice. Coinjection of 3 mg/kg (i.v.) SR141716A, a CB1 receptor antagonist, withR‐[125I]AM2233 inhibited the radiotracer binding almost to nonspecific levels in the striatum, globus pallidus, and substantia nigra, although residual binding to a non‐CB1 receptor remained in the hippocampus. In contrast to the effect of SR141716A, coinjection of 10 mg/kg (i.v.) WIN55212‐2, a high dose that produced an immediate and profound immobility and catalepsy in the mice, reduced CB1 receptor‐specific binding ofR‐[125I]AM2233 in CB1 receptor‐rich areas by only 21–43%. These observations suggest that the behavioral effects of CB1 receptor agonists are manifested with a relatively small fraction of the agonist‐preferring affinity state of the receptor occupied. Synapse 60:93–101, 2006. © 2006 Wiley‐Liss, Inc.