An alpha 2-adrenergic receptor subtype has been isolated from a human genomic spleen library using the human 5-hydroxytryptamine1A receptor gene (also known as G-21) as a probe. This adrenergic receptor gene encodes a protein of 450 amino acids and does not contain any consensus sequences for N-linked glycosylation in its amino terminus or extracellular loops. This receptor is also distinguished by the presence of 12 consecutive glutamic acid residues in the region of its third intracellular loop. The deduced amino acid sequence shows greatest homology to previously cloned human alpha 2-adrenergic receptors and has structural similarities to other guanine nucleotide-binding protein-coupled receptors. The DNA encoding the human alpha 2 receptor was stably transfected into mouse fibroblast Ltk- cells and radioligand binding studies were performed using the alpha 2 antagonist [3H]rauwolscine. [3H]Rauwolscine bound with high affinity (Kd = 0.33 nM) and in a saturable manner (Bmax = 1.4 pmol/mg of protein). Pharmacological characterization of this receptor indicated a rank order of potency of yohimbine greater than prazosin greater than oxymetazoline. Additionally, 100 microM 5'-guanylylimidodiphosphate, produced a rightward shift in the epinephrine competition curve, with resultant increases in both the Ki value and Hill coefficient, suggestive of a functional interaction of the cloned receptor with native guanine nucleotide-binding protein(s) of Ltk- membranes. The data presented here are consistent with previous biochemical and pharmacological studies on alpha 2 receptors and are supportive of the designation of this receptor as an alpha 2B subtype.