The present study was conducted to characterise the centrally active analgesic drug tramadol hydrochloride [(1RS,2RS)-2-[(dimethyl-amino)-methyl]-1-(3-methoxyphenyl)-cyclohe xanol hydrochloride] and its metabolites M1, M2, M3, M4 and M5 at the cloned human mu-opioid receptor. Membranes from stably transfected Chinese hamster ovary (CHO) cells were used to determine the four parameters of the ligand-receptor interaction: the affinity of (+/-)-tramadol and its metabolites was determined by competitive inhibition of [3H]naloxone binding under high and low salt conditions. The agonist-induced stimulation of [35S]GTPgammaS binding permits the measurement of potency (EC50), efficacy (Emax = maximal stimulation) and relative intrinsic efficacy (effect as a function of receptor occupation). The metabolite (+)-M1 showed the highest affinity (Ki=3.4 nM) to the human mu-opioid receptor, followed by (+/-)-M5 (Ki=100 nM), (-)-M1 (Ki=240 nM) and (+/-)-tramadol (Ki=2.4 microM). The [35S]GTPgammaS binding assay revealed an agonistic activity for the metabolites (+)-M1, (-)-M1 and (+/-)-M5 with the following rank order of intrinsic efficacy: (+)-M1>(+/-)-M5>(-)-M1. The metabolites (+/-)-M2, (+/-)-M3 and (+/-)-M4 displayed only weak affinity (Ki> 10 microM) and had no stimulatory effect on GTPgammaS binding. These data indicate that the metabolite (+)-M1 is responsible for the mu-opioid-derived analgesic effect.