The functioning of heterotrimeric G protein α subunits in the transduction of hormonal signals to appropriate intracellular responses is well recognized. Much less is known about the distribution of isoforms and functions of G protein β subunits. Here, using specific antibodies, we documented that in plasma membranes of the thyroid cell line Nthy-ori 3-1 all Gβ isoforms-Gβ1, Gβ2, Gβ3, Gβ4 and Gβ5 are present, while the Gβ3 occurs in minute amount. In plasma membrane fraction isolated from pooled postoperative thyroids of patients with nodular goiter and Graves’ disease, the Gβ1, Gβ2, Gβ4 and Gβ5 subunits were found, whereas Gβ3 could not be detected.Competition studies revealed that the Gβ2 is the principal Gβ subunit in membranes from cultured thyroid cells, originated from normal thyroid, as well as in membranes from patients’ thyroids. This suggests that Gβ2 subunit cooperates with Gαs subunit, the most active of the Gα variants, during stimulation of adenylate cyclase which constitutes the main route of physiological thyroid stimulation.