We previously reported that in the pregnant human myometrium the binding sites labeled with [3H]idazoxan have the pharmacological characteristics of alpha 2-adrenergic receptors. Competitive experiments have also revealed that the stable guanine nucleotide analog guanyl-5'-imidodiphosphate decreases the apparent affinity of norepinephrine and clonidine for myometrial [3H]idazoxan-binding sites. In the present study, the alpha 2-adrenergic mechanism in this tissue was further approached by measuring adenylate cyclase responses and examining the different pertussis toxin-sensitive G-proteins. The two alpha 2-adrenergic agonists norepinephrine and clonidine inhibited adenylate cyclase activity in both the outer and inner layers of the pregnant human myometrium. The inhibitory effect of these agonists is completely reversed by alpha 2-adrenergic antagonists such as yohimbine and idazoxan. Pretreatment with pertussis toxin completely suppresses the inhibition of adenylate cyclase mediated by alpha 2-adrenergic receptors, suggesting that an inhibitory protein of the Gi type is involved. Pertussis toxin, known to catalyze the ADP ribosylation of the alpha-subunit of several G-proteins, labels three substrates at 39, 40, and 41 kDa. The more intense labeling occurring on the 40- to 41-kDa components are assigned to alpha-subunits of Gi-like proteins, whereas that at 39 kDa might correspond to a Go alpha-like substrate. These results indicate the presence of alpha 2-adrenergic receptors in the human myometrium at the end of pregnancy that are functionally linked to inhibition of adenylate cyclase activity via the Gi protein.