Peptidergic nerve fibers provide important contributions to urethral function. Urethral innervation of female mice is not well documented.

To determine the distribution and projection sites of nerve fibers immunoreactive for vasoactive intestinal peptide (VIP), calcitonin gene‐related peptide (CGRP), substance P (SP), and neuropeptide Y (NPY) in the urethra of wild‐type control mice and compare innervation characteristics between the proximal and distal urethra of young nullipara and older multipara mice. Furthermore, to identify the location and neurochemical coding of the spinal afferent nerve endings in the urethra, whose sensory neurons reside in lumbosacral dorsal root ganglia (DRG).

Multiple labeling immunohistochemistry of urethral sections of nulliparous (6‐8 weeks old), and multiparous (9‐12 months old) mice, and anterograde axonal tracing from L5‐S2 (DRG) in vivo.

Abundant VIP‐, CGRP‐, SP‐, and NPY‐immunoreactive nerve fibers were identified in the adventitia, muscularis, and lamina propria of proximal and distal segments of the urethra. A proportion of fibers were closely associated with blood vessels, glands, and cells immunoreactive for PGP9.5. The epithelium contained abundant nerve fibers immunoreactive for CGRP and/or SP. Epithelial innervation was increased in the distal urethra of multipara mice. Abundant fibers were traced from L5‐S2 DRG to all urethral regions.

We present the first identification of spinal afferent endings in the urethra. Peptidergic nerve fibers, including multiple populations of spinal afferents, provide rich innervation of the female mouse urethra. The morphology of fibers in the epithelium and other regions suggests multiple nerve‐cell interactions impacting on urethral function.