Abstract Neurturin (NRTN) is a neurotrophic factor required for the development of many parasympathetic neurons and normal cholinergic innervation of the heart, lacrimal glands and numerous other tissues. Previous studies with transgenic mouse models showed that NRTN is also essential for normal development and function of the retina (J. Neurosci. 28:4123–4135, 2008). NRTN knockout (KO) mice exhibit a marked thinning of the outer plexiform layer (OPL) of the retina, with reduced abundance of horizontal cell dendrites and axons, and aberrant projections of horizontal cells and bipolar cells into the outer nuclear layer. The effects of NRTN deletion on specific neurotransmitter systems in the retina and on cholinergic innervation of the iris are unknown. To begin addressing this deficiency, we used immunohistochemical methods to study cholinergic and noradrenergic innervation of the iris and the presence and localization of cholinergic and dopaminergic neurons and nerve fibers in eyes from adult male wild-type (WT) and NRTN KO mice (age 4–6 months). Mice were euthanized, and eyes were removed and fixed in cold neutral buffered formalin or 4% paraformaldehyde. Formalin-fixed eyes were embedded in paraffin, and 5 μm cross-sections were collected. Representative sections were stained with hematoxylin and eosin or processed for fluorescence immunohistochemistry after treatment for antigen retrieval. Whole mount preparations were dissected from paraformaldehyde fixed eyes and used for immunohistochemistry. Cholinergic and catecholaminergic nerve fibers were labeled with primary antibodies to the vesicular acetylcholine transporter (VAChT) and tyrosine hydroxylase (TH), respectively. Cholinergic and dopaminergic cell bodies were labeled with antibodies to choline acetyltransferase (ChAT) and TH, respectively. Cholinergic innervation of the mouse iris was restricted to the sphincter region, and noradrenergic fibers occurred throughout the iris and in the ciliary processes. This pattern was unaffected by deletion of NRTN. Furthermore, functional experiments demonstrated that cholinergic regulation of the pupil diameter was retained in NRTN KO mice. Hematoxylin and eosin stains of the retina confirmed a marked thinning of the OPL in KO mice. VAChT and ChAT staining of the retina revealed two bands of cholinergic processes in the inner plexiform layer, and these were unaffected by NRTN deletion. Likewise, NRTN deletion did not affect the abundance of ChAT-positive ganglion and amacrine cells. In marked contrast, staining for TH showed an increased abundance of dopaminergic processes in the OPL of retina from KO mice. Staining of retinal whole mounts for TH showed no difference in the abundance of dopaminergic amacrine cells between WT and KO mice. These findings demonstrate that the neurotrophic factor NRTN is not required for the development or maintenance of cholinergic innervation of the iris, cholinergic control of pupil diameter, or for development of cholinergic and dopaminergic amacrine cells of the retina. However, NRTN deficiency causes a marked reduction in the size of the OPL and aberrant growth of dopaminergic processes into this region.