Previous studies have established that the human colon carcinoma cell line HT29 expresses an alpha 2-adrenergic receptor of the alpha 2A subtype, which is negatively coupled to adenylate cyclase. The purpose of the present study was to examine the mechanisms of alpha 2-adrenergic signal transduction in these cells. [32P]ADP-ribosylation with pertussis toxin and immunoblots using antibodies specific for the Gi alpha-subunits indicated that two distinct Gi-proteins (Gi2 and Gi3) were present in HT29-cell membranes. Treatment of intact cells with pertussis toxin resulted in a time-dependent decrease in the amount of [32P]ADP-ribosylatable Gi2 and Gi3, which coincided with a diminution in the number of alpha 2-adrenergic receptors in high-affinity state for agonists and with a progressive loss of ability of UK14304 to inhibit forskolin-stimulated accumulation of cyclic AMP. When membranes were [32P]ADP-ribosylated with cholera toxin in the absence of exogenous added guanine nucleotides, radioactivity was incorporated into a 45 kDa polypeptide representing Gs, as well as into 40-41 kDa polypeptides corresponding to Gi3 and Gi2. The amount of radioactivity incorporated into the two GiS under basal conditions was decreased by addition of the alpha 2-antagonist RX821002. It was not significantly affected by addition of clonidine (partial alpha 2-agonist), but was doubled by the addition of UK14304 (full alpha 2-agonist). This effect was blocked by RX821002. Study of adenylate cyclase activity indicated that preincubation of HT29 membranes with the antibody AS/7 (anti-alpha i1/alpha i2), but not with the antibody EC/2 (anti-alpha i3), attenuated the inhibitory effect of UK14304 on forskolin-stimulated adenylate cyclase. These data demonstrate that the alpha 2A-adrenergic receptor is coupled to both Gi2 and Gi3, and identify Gi2 as the major mediator of inhibition of adenylate cyclase in HT29 cells.