These studies examined changes in the pituitary adenylate cyclase activating polypeptide (PACAP) expression in micturition reflex pathways after spinal cord injury (SCI) of various durations. In spinal-intact animals, PACAP immunoreactivity (IR) was expressed in fibers in the superficial dorsal horn in all segmental levels examined (L1, L2, L4-S1). Bladder-afferent cells (35-45%) in the dorsal root ganglia (DRG; L1, L2, L6, S1) from spinal-intact animals also exhibited PACAP-IR. After SCI (6 weeks), PACAP-IR was dramatically increased in spinal segments and DRG (L1, L2, L6, S1) involved in micturition reflexes. The density of PACAP-IR was increased in the superficial laminae (I-II) of the L1, L2, L6, and S1 spinal segments. No changes in PACAP-IR were observed in the L4-L5 segments. Staining was also dramatically increased in a fiber bundle extending ventrally from Lissauer's tract (LT) in lamina I along the lateral edge of the dorsal horn to the sacral parasympathetic nucleus (SPN) in the L6-S1 spinal segments (lateral collateral pathway of Lissauer, LCP). After SCI (range 48 h to 6 weeks), PACAP-IR in cells in the L1, L2, L6, and S1 DRG significantly (P < or = 0.001) increased and the percentage of bladder-afferent cells expressing PACAP-IR also significantly (P < or = 0.001) increased (70-92%). No changes were observed in the L4-L5 DRG. PACAP-IR was reduced throughout the urothelium and detrusor smooth muscle whole mounts after SCI. These studies demonstrate changes in PACAP expression in micturition reflex pathways after SCI that may contribute to urinary bladder dysfunction or reemergence of primitive voiding reflexes after SCI.