The purpose of the present study was to ascertain the role of adenylate (AC) versus guanylate cyclase (GC) signaling pathways in the internal anal sphincter (IAS) smooth muscle relaxation by ␤ 1 -, ␤ 2 -, and ␤ 3 -adrenoceptor (AR) activation by xamoterol, procaterol, and disodium 5-[(2R)-2-(3-chlorophenyl)-2-hydroxy-ethyl]amino)propyl]-1,3-benzodioxole-2,2-dicarboxylate (CL 316243), respectively. The above-mentioned agonists produced concentration-dependent relaxation of the smooth muscle strips. Both the selective G i/o ␣ and G s ␣ antagonists 8,8Ј-(carbonylbis(imino-3,1-phenylene))bis-(1,3,5-naphthalene trisulfonic acid) (NF 023) and 4,4Ј,4Љ,4ٞ-(carbonylbis(imino-5,1,3-benzenetriylbis(carbonylimino)))tetrakisbenzene-1,3-disulfonic acid (NF 449), respectively, inhibited the relaxation induced by procaterol. However, only NF 023 inhibited the relaxation induced by xamoterol and CL 316243. 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one, a soluble GC inhibitor, significantly inhibited the relaxation induced by different agonists. In contrast, the selective AC inhibitor [9-(tetrahydro-2Ј-furyl)adenine] (SQ 22536) inhibited only the relaxation induced by procaterol. (9R,10S,12S)-2,3,9,10,11,12-Hexahydro-10-hydroxy-9-methyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3-fg: 3Ј,2Ј,1Ј-kl]pyrrolo[3,4-l][1,6]benzodiazocine-10-carboxylic acid, hexyl ester (KT 5720), a cAMP-dependent protein kinase inhibitor, attenuated the relaxation by procaterol, whereas (9S,10R,12R)-2,3,9,10,11,12, hexahydro-10-methoxy-2,9-dimethyl-1-oxo-9.12-epoxy-1H-diindolo[1,2,3-fg:3Ј,2Ј,1Ј-kl]pyrrolo [3,4-I] [1,6]benzodiazocine-10-carboxylic acid methyl ester (KT 5823), a selective cGMP-dependent protein kinase (PKG) inhibitor, attenuated the relaxation induced by xamoterol and CL 316243. Xamoterol produced significant increase in cGMP levels, whereas only procaterol enhanced the cAMP levels. Western blot analysis confirmed the presence of ␤ 1 , ␤ 2 , and ␤ 3 -AR subtypes in the IAS. In summary, ␤ 2 -AR activates both G s ␣ and G i/o ␣-protein subunits and induces relaxation in the rat IAS via both cAMP/cGMP pathways. In contrast, the ␤ 1 /␤ 3 -ARs activation causes the smooth muscle relaxation via G i/o ␣-protein subunit/GC/GMP/PKG pathway. These studies are important for the understanding of intracellular mechanisms underlying IAS smooth muscle relaxation and in turn the pathophysiology of certain anorectal motility disorders.The internal anal sphincter (IAS) is an involuntary muscle that maintains a state of spontaneous tonic contraction and is responsible for anorectal continence, and its relaxation, on the other hand, is critical for the anorectal inhibitory reflex. However, under certain conditions, the increase in the intraluminal pressure of the anal canal results in anorectal dysfunctions such as anal fissures, constipation, hemorrhoids, and Hirschsprung's disease (Cook et al., 2001;Azpiroz and Whitehead, 2002). Consequently, there is an increasing interest in the agents that cause selective, potent, and