We and others have recently demonstrated that the pharmacological tolerance observed after prolonged exposure to plant and synthetic cannabinoids in adult individuals seems to have a pharmacodynamic basis, based on the observed down-regulation of cannabinoid receptors in the brain of cannabinoid-tolerant rats. However, we were unable to elicit a similar receptor down-regulation after a chronic exposure to anandamide, the first discovered endogenous cannabinoid, possibly because of its rapid metabolic breakdown in arachidonic acid and ethanolamine. The present study was designed to progress in these previous studies, by using R-methanandamide, a more stable analog, instead anandamide. In addition, we examined not only cannabinoid receptor binding, but also WIN-55,212-2-stimulated [35S]-GTPgammaS binding, by autoradiography, and cannabinoid receptor mRNA levels, by in situ hybridization. Results were as follows. The daily administration of R-methanandamide for a period of five days produced decreases in cannabinoid receptor binding in the lateral caudate-putamen, cerebellum, entopeduncular nucleus and substantia nigra. The remaining areas, the medial caudate-putamen, globus pallidus, cerebral cortex (layers I and VI), hippocampus (dentate gyrus and Ammon's horn) and several limbic structures (nucleus accumbens, septum nuclei and basolateral amygdaloid nucleus), exhibited no changes in cannabinoid receptor binding. Similarly, the levels of cannabinoid receptor mRNA expression decreased in the lateral and medial caudate-putamen and in the CA1 and CA2 subfields of the Ammon's horn in the hippocampus after the chronic exposure to R-methanandamide, whereas the remaining areas showed no changes. WIN-55,212-2-stimulated [35S]-GTPgammaS binding did not change in the lateral caudate-putamen, cerebral cortex (layer I), septum nuclei and hippocampal structures (dentate gyrus and Ammon's horn) of animals chronically exposed to R-methanandamide, whereas a certain trend to decrease could be observed in the substantia nigra and deep layer (VI) of the cerebral cortex in these animals. In summary, as reported for other cannabinoid receptor agonists, the prolonged exposure of rats to R-methanandamide, a more stable analog of anandamide, was able to produce cannabinoid receptor-related changes in contrast with the absence of changes observed early with the metabolically labile anandamide. The observed changes exhibited an evident regional pattern with areas, such as basal ganglia, cerebellum and hippocampus, responding to chronic R-methanandamide treatment while regions, such as the cerebral cortex and limbic nuclei, not responding.