Functional and molecular approaches were used to characterize the β-AR subtypes mediating relaxation of rat ileal smooth muscle.In functional studies, (−)-isoprenaline relaxation was unchanged by CGP20712A (β1-AR antagonist) or ICI118551 (β2-AR antagonist) but shifted by propranolol (pKB=6.69). (±)-Cyanopindolol, CGP12177 and ICID7114 did not cause relaxation but antagonized (−)-isoprenaline relaxation.BRL37344 (β3-AR agonist) caused biphasic relaxation. The high affinity component was shifted with low affinity by propranolol, (±)-cyanopindolol, tertatolol and alprenolol. CL316243 (β3-AR agonist) relaxation was unaffected by CGP20712A or ICI118551 but blocked by SR58894A (β3-AR antagonist; pA2=7.80). Enhanced relaxation after exposure to forskolin and pertussis toxin showed that β3-AR relaxation can be altered by manipulation of components of the adenylate cyclase signalling pathway.The β1-AR agonist RO363 relaxed the ileum (pEC50=6.18) and was blocked by CGP20712A. Relaxation by the β2-AR agonist zinterol (pEC50=5.71) was blocked by SR58894A but not by ICI118551.In rat ileum, β1-, β2- and β3-AR mRNA was detected. Comparison of tissues showed that β3-AR mRNA expression was greatest in WAT>colon=ileum>cerebral cortex>soleus; β1-AR mRNA was most abundant in cerebral cortex>WAT>ileum=colon>soleus; β2-AR mRNA was expressed in soleus>WAT>ileum=colon>cerebral cortex.These results show that β3-ARs are the predominant β-AR subtype mediating rat ileal relaxation while β1-ARs may produce a small relaxation. The β2-AR agonist zinterol produces relaxation through β3-ARs and there was no evidence for the involvement of β2-ARs in relaxation despite the detection of β2-AR mRNA.