The stress-related neuropeptide, corticotropin-releasing factor (CRF), is prominent in neurons of the pontine micturition center, Barrington’s nucleus. These neurons co-innervate spinal preganglionic neurons that control the bladder, and locus coeruleus (LC) neurons that provide norepinephrine innervation throughout the brain. Adeno-associated viral (AAV) vector-mediated transfer of CRF cDNA was used to increase CRF expression in Barrington’s nucleus neurons and investigate the impact of a gain of function in Barrington’s nucleus spinal and LC projections. AAV transfer of the reverse CRF cDNA sequence served as the control. Bladder urodynamics and behavior were assessed 4 weeks after vector injection into Barrington’s nucleus. Rats with bilateral injections of AAV–CRF cDNA into Barrington’s nucleus had immunohistochemical evidence of CRF overexpression in neurons and transport to the spinal cord and LC. The bladder : body weight ratio was greater and micturition pressure was less in these rats compared with controls, consistent with an inhibitory influence on bladder function. Other indices of urodynamic function were not altered. CRF innervation of the LC was increased in rats with bilateral Barrington’s nucleus injections of AAV–CRF cDNA, and this was associated with increased burying behavior, an endpoint of LC activation by CRF. The results provide immunohistochemical evidence for viral vector-induced CRF overexpression in Barrington’s nucleus neurons and underscore the ability of AAV vector-mediated transfer to increase CRF function in selective circuits. The findings support an inhibitory influence of CRF in Barrington’s nucleus regulation of the bladder and an excitatory influence on the brain norepinephrine system that translates to behavioral activation.