To determine the functional role of M(3) and M(5) muscarinic acetylcholine receptor subtypes in ophthalmic arteries using gene-targeted mice.Muscarinic receptor gene expression was quantified in murine ophthalmic arteries using real-time PCR. To test the functional relevance of M(3) and M(5) receptors, ophthalmic arteries from mice deficient in either subtype (M3R(-/-), M5R(-/-), respectively) and wild-type controls were isolated, cannulated with micropipettes, and pressurized. Changes in luminal vessel diameter in response to muscarinic and nonmuscarinic receptor agonists were measured by video microscopy.With the use of real-time PCR, all five muscarinic receptor subtypes were detected in ophthalmic arteries. However, mRNA levels of M(1), M(3), and M(5) receptors were higher than those of M(2) and M(4) receptors. In functional studies, after preconstriction with phenylephrine, acetylcholine and carbachol produced concentration-dependent dilations of ophthalmic arteries that were similar in M5R(-/-) and wild-type mice. Strikingly, cholinergic dilation of ophthalmic arteries was almost completely abolished in M3R(-/-) mice. Deletion of either M(3) or M(5) receptor did not affect responses to nonmuscarinic vasodilators such as bradykinin or nitroprusside.These findings provide the first evidence that M(3) receptors are critically involved in cholinergic regulation of diameter in murine ophthalmic arteries.