Chinese hamster ovary (CHO) cells stably transfected to express different densities of the human alpha2A-, alpha2B- and alpha2C-adrenoceptor subtypes, were used to characterize the regulation of adenylyl cyclase activity by alpha2-adrenoceptor agonists. In isolated cell membranes, activation of alpha2A- and alpha2C-adrenoceptors did not affect basal enzyme activity, but activation of alpha2B-adrenoceptors stimulated adenylyl cyclase activity. The extent of stimulation was dependent on the receptor density and was insensitive to pertussis toxin treatment. In the presence of 10 microM forskolin all three receptor subtypes mediated inhibition of adenylyl cyclase activity in a pertussis toxin-sensitive manner. In experiments performed with intact cells the same pattern could be seen: the basal production of cAMP was not affected when alpha2C-adrenoceptors were activated, but activated alpha2B-adrenoceptors mediated stimulation of cAMP production. In the presence of forskolin, both receptor subtypes mediated inhibition of cAMP production. Our results suggest that alpha2B-adrenoceptors are coupled to both Gi and Gs proteins. The signal transduction pathway to which the receptor is coupled is not dependent on receptor density, but its effect on adenylyl cyclase regulation is dependent on the current activity of adenylyl cyclase. The results also suggest that the alpha2A- and alpha2C-subtypes are preferentially coupled to Gi and transduce only inhibition of adenylyl cyclase activity in transfected CHO cells. At low densities of alpha2C-adrenoceptors, clonidine was a partial agonist, but in clones expressing high levels of alpha2C-adrenoceptors, clonidine acted as a full agonist by inhibiting cAMP accumulation with the same efficacy as (-)-noradrenaline. This demonstrates that receptor reserve can mask partial agonist activity.