The purpose of the present study was to characterize different beta-adrenoceptors (beta-ARs) and determine their role in the spontaneously tonic smooth muscle of the internal anal sphincter (IAS). The beta-AR subtypes in the opossum IAS were investigated by functional in vitro, radioligand binding, Western blot, and reverse transcription-polymerase chain reaction (RT-PCR) studies. ZD 7114 [(S)-4-[2-hydroxy-3-phenoxypropylaminoethoxy]-N-(2-methoxyethyl)phenoxyacetamide], a selective beta(3)-AR agonist, caused a potent and concentration-dependent relaxation of the IAS smooth muscle that was antagonized by the beta(3)-AR antagonist SR 59230A [1-(2-ethylphenoxy)-3-[[(1S)-1,2,3,4-tetrahydro-1-naphthalenyl]amino]-(2S)-2-propanol hydrochloride]. Conversely, the IAS smooth muscle relaxation caused by beta(1)- and beta(2)-AR agonists (xamoterol and procaterol, respectively) was selectively antagonized by their respective antagonists CGP 20712 [(+/-)-2-hydroxy-5-[2-[[2-hydroxy-3-[4-[1-methyl-4-(trifluoromethyl)-1H-imidazol-2-yl]phenoxy]propyl]amino]ethoxy]-benzamide methanesulfonate salt] and ICI 118551. Saturation binding of [(125)I]iodocyanopindolol to beta-AR subtypes revealed the presence of a high-affinity site (K(d1) = 96.4 +/- 8.7 pM; B(max1) = 12.5 +/- 0.6 fmol/mg protein) and a low-affinity site (K(d2) = 1.96 +/- 1.7 nM; B(max2) = 58.7 +/- 4.3 fmol/mg protein). Competition binding with selective beta-AR antagonists revealed that the high-affinity site correspond to beta(1)/beta(2)-AR and the low affinity site to beta(3)-AR. Receptor binding data suggest the predominant presence of beta(3)-AR over beta(1)/beta(2)-AR. Western blot studies identified beta(1)-, beta(2)-, and beta(3)-AR subtypes. The presence of beta(1)-, beta(2)-, and beta(3)-ARs was further demonstrated by mRNA analysis using RT-PCR. The studies demonstrate a comprehensive functional and molecular characterization of beta(1)-, beta(2)-, and beta(3)-ARs in IAS smooth muscle. These studies may have important implications in anorectal and other gastrointestinal motility disorders.