Stable S115 mouse mammary tumour cell lines, expressing separately alpha 2A-C10, alpha 2B-C2 and alpha 2C-C4 adrenoceptors were used to compare the receptor binding properties of alpha 2-adrenoceptor agonists with their potency in inhibiting cAMP production. All tested agonists detected high and low affinity binding sites in all three receptor subtypes. In the presence of the GTP analogue Gpp(NH)p (10 microM), all displacement curves were shifted to the right and were best modelled by one-site fits, suggesting that the receptor subtypes are coupled to G-proteins. The extent of the Gpp(NH)p-induced shift was greatest in the alpha 2A-C10 subtype, smaller in alpha 2C-C4, and minimal in alpha 2B-C2. All three receptor subtypes were also coupled to inhibition of forskolin-stimulated cAMP production through pertussis toxin-sensitive G-proteins. For the full agonists noradrenaline, UK 14,304, and dexmedetomidine, the maximal inhibitory effect on cAMP production was smaller in the alpha 2B-C2 subtype (35%) than in the alpha 2A-C10 and alpha 2C-C4 subtypes (50-70%). After treatment of cells expressing alpha 2B-C2 receptors with pertussis toxin, cAMP production was increased by up to 58% by alpha 2-adrenoceptor agonists. Similar stimulation of adenylyl cyclase activity could not be demonstrated at the other two receptor subtypes. In conclusion, these results demonstrate that (1) alpha 2-adrenoceptor agonists may be characterized by an agonist-type binding pattern in homogenates of transfected S115 cells, (2) all three alpha 2-adrenoceptor subtypes are coupled to inhibition of adenylyl cyclase in S115 cells through pertussis toxin-sensitive G-proteins, (3) the receptor-effector coupling in S115 cells is different among the subtypes so that the alpha 2A-C10 subtype is coupled with high efficacy but with low sensitivity, the alpha 2B-C2 subtype with low efficacy but high sensitivity, and the alpha 2C-C4 subtype with both high efficacy and high sensitivity, and (4) at least alpha 2B-C2 receptors may also be coupled to stimulation of adenylyl cyclase activity, presumably through Gs.