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The α-adrenergic receptors (α-ARs) in canine mesenteric vein (DMV) were studied by using nonselective agonists and selective antagonists in functional studies and in ligand binding to classify the subtypes present. Based on functional studies of phenylephrine (PE)-induced contractions and ligand-binding interactions of [3H]-prazosin with prazosin (PR), WB 4101 (WB), 5-methylurapidil (5-MU), BMY 7378, and SK&F 105854, and pretreatment with chloroethylclonidine (CEC), DMV α1-ARs resembled the α1D subtype. However, the affinity of PR assessed in functional and ligand-binding studies was less (pKB,D,i ≤ 9) than expected from previous characterization of cloned rat or human α1-AR (pKi ≥ 10). Interactions with 5-MU, BMY 7378, or SK&F 105854 suggested the presence of some α1-ARs that were not typical of α1D-AR and that binding and functional interactions did not yield corresponding results. PR binding was abolished by treatment with CEC, contractile responses to PE were reduced in Emax, and the concentration-effect curve shifted to the left, as previously reported. DMVs contracted in response to α2-AR agonists and were studied when contractions were potentiated by increasing extracellular KCl to 20 mM. Rauwolscine (RAU) had KB values at these sites consistent with KD values in binding studies. CEC had no effect on RAU binding in DMV. Ligand-binding studies to [3H]-RAU sites did not reveal a clear identification of subtype, but these α2-ARs were clearly not α2B-ARs. We conclude that canine mesenteric vein contains α1D-like ARs, but with significant differences, and an unclassifiable α2-AR. There may also be a smaller population of other, not α1D-like ARs, receptors, mediating responses to PE and binding of prazosin.