Many hormones and neurotransmitters bind to membrane-bound receptors that are coupled to signal generating enzymes or ion channels via signal transducing GTP-binding proteins termed G proteins. Although receptors and second messengers have been extensively studied in cells of the respiratory system, the G proteins responsible for the coupling of these proteins have not been well-characterized. Therefore, we used immunoblot analysis to determine expression of G protein alpha and beta subunits in membranes prepared from cells and tissues of the respiratory system, including cultured canine tracheal epithelium, cleanly dissected canine tracheal smooth muscle, canine large conducting airways, and canine and human lung parenchyma. The two isoforms of Gs alpha (45 and 52 kDa) were present in all tissues, with a predominant expression of the 45 kDa isoform. Plasma membranes prepared from canine tracheal epithelium and muscle, and human lung parenchyma, contained greater amounts of Gs alpha than membranes prepared from canine bronchus and lung. Relative levels of immunoreactive G(i) alpha(2), G(i) alpha(3), Gq/G11 alpha, beta 1 and beta 2 were similar in all of the tissues studied. By contrast, G(o) alpha was absent in cultured tracheal epithelium, and tracheal smooth muscle expressed greater amounts of G(i) alpha(2) compared to G(i) alpha(3). Specificity of G protein expression can provide one regulatory mechanism for functional biochemical pathways within cells. The demonstration of specific G protein subunits is the first step in the molecular characterization of the regulation of these pathways, both in normal tissues and in disease states.