We have investigated the pharmacology of two central human cannabinoid receptor isoforms, designated CB1 and CB1A, stably expressed in Chinese hamster ovary cell lines, designated as CHO-CB1 and CHO-CB1A, respectively. In direct binding assays on isolated membranes the agonist [3H]CP 55,940 bound in a saturable and highly specific manner to both cannabinoid receptor isoforms. Competition binding experiments performed with other commonly used receptor agonists showed the following rank order of potency: CP 55,940 > tetrahydrocannabinol > WIN 55212-2 > anandamide. Except for the endogenous ligand anandamide (CB1, Ki = 359.6 nM vs. CB1A, Ki = 298 nM), these agonists bound to CB1A (CP 55,940, WIN 55212-2 and delta 9-THC, Ki = 7.24,345 and 26.7 nM, respectively) with about 3-fold less affinity than to CB1 (CP 55,940, WIN 55212-2 and delta 9-THC, Ki = 2.26, 93 and 7.1 nM, respectively). The cannabinoid receptor antagonist SR 141716A also bound to CB1A (Ki = 43.3 nM) with slightly less affinity than to CB1 (Ki = 4.9 nM). Cannabinoid receptor-linked second messenger system studies performed in the CHO-CB1 and CHO-CB1A cells showed that both receptors mediated their action through the agonist-induced inhibition of forskolin-stimulated cAMP accumulation. This activity was totally blocked by pretreatment with PTX. Additionally, both isoforms activated mitogen-activated protein kinase. The selective antagonist SR 141716A was able to selectively block these responses in both cell lines, to an extent that reflected its binding characteristics. Our results show that the amino-truncated and -modified CB1 isoform CB1A exhibits all the properties of CB1 to a slightly attenuated extent.