To determine the intracellular signal transduction pathway responsible for the development of tolerance/dependence, the ability of Gzalpha to substitute for pertussis toxin (PTX)-sensitive G proteins in mediating adenylyl cyclase (AC) supersensitivity was examined in the presence of defined AC isoforms. In transiently micro-opioid receptor (OR) transfected COS-7 cells (endogenous inhibitory G proteins: Gialpha2, Gialpha3 and Gzalpha), neither acute (1 micro mol/L) nor chronic morphine treatment (1 micromol/L; 18 h) influenced intracellular cAMP production. Coexpression of the micro -OR together with AC type V and VI fully restored the ability of morphine to acutely inhibit cAMP generation. Chronic morphine treatment further resulted in the development of tolerance/dependence, as assessed by desensitization of the acute inhibitory opioid effect (tolerance) as well as the induction of AC supersensitivity after drug withdrawal (dependence). Specific direction of micro -OR signalling via Gzalpha by both PTX treatment and Gzalpha over-expression had no effect on chronic morphine regulation of AC type V, but completely abolished the development of tolerance/dependence with AC type VI. Similar results were obtained in stably micro -OR-expressing HEK293 cells transiently cotransfected with Gzalpha and either AC type V or VI. Coprecipitation studies further verified that Gzalpha specifically binds to AC type V but not type VI. Taken together, these results demonstrate that in principle each of the OR-activated G proteins per se is able to mediate AC supersensitivity. However, they also indicate that it is the molecular nature of AC isoform that selects and determines the OR-activated G protein mediating tolerance/dependence.