Objective: Muscarinic acetylcholine receptors, consisting of five subtypes (M1-M5), participate in numerous physiological processes in the eye, such as regulation of pupil size and intraocular pressure. Recently, we reported that M3 receptors mediate cholinergic responses in the murine ophthalmic artery. The purpose of this study was to identify the physiological actions in the eye that are influenced by M3 receptors and to examine, whether this subtype is involved in glaucoma development. Methods: Experiments were performed in mice deficient in M3 receptors (M3R-/-) and age- matched wild-type controls. Pupil diameter was assessed from photographs taken under standardized illumination. To measure intraocular pressure, a handheld tonometer (Tono- PenXL®) was used. Using real-time PCR, muscarinic receptor gene expression was quantified in isolated retinal arterioles of wild-type mice. Responses of retinal arterioles to cholinergic agents were measured in isolated retinae using videomicroscopy. Moreover, retinal ganglion cell density was determined by counting cells in the ganglion cell layer of flat-mounted retinae stained with cresyl violet. Results: Pupil diameter was markedly increased in M3R-/- mice compared to their wild-type controls (1.79±0.10 mm and 0.45±0.02 mm; M3R-/- vs. wild-type mice; P<0.0001). Using real-time PCR, only M3 receptor mRNA was detected in isolated retinal arterioles of wild-type mice. In line with this finding, responses of retinal arterioles to acetylcholine were abolished in M3R-/- mice, while dose-dependent dilation was observed in wild-type mice (2±2% and 14±3% at 100 mM; M3R-/- vs. wild-type mice; P<0.01). Intraocular pressure and retinal ganglion cell density did not differ between M3R-/- and wild- type mice. Conclusions: These findings provide evidence that M3 receptors are involved in cholinergic regulation of pupil and retinal arteriole diameter. However, M3 receptors do not appear to be involved in glaucoma development.