A number of in vitro studies suggest that many important developmental and functional events in the enteric nervous system are regulated by the intracellular signaling enzyme cAMP protein kinase A (PKA). To evaluate the in vivo significance of these observations, a Cre-inducible, dominant-negative, mutant regulatory subunit (RIalphaB) of PKA was activated in enteric neurons by either a Proteolipid protein-Cre transgene or a Hox11L1-Cre "knock-in" allele. In both models, RIalphaB activation resulted consistently in profound distension of the proximal small intestine within 2 weeks after birth. Intestinal transit of radio-opaque tracers was severely retarded in the double-transgenic animals, which died shortly after weaning. In the enteric nervous system, recombination was restricted to neurons as demonstrated by histochemical analysis and confocal microscopic colocalization of a Cre recombinase-dependent reporter gene with the neuronal marker Hu(C/D), in contrast with the glial marker S100. Histochemical analysis of beta-galactosidase expression and acetylcholinesterase activity, as well as neuronal counts, demonstrated that intestinal dysmotility was not associated with obvious malformation of the myenteric plexus. However, inhibition of PKA activity in enteric neurons disrupted the major motor complexes of isolated intestinal segments in vitro. These results provide strong evidence that PKA activity plays a critical role in enteric neurotransmission in vivo, and highlight neuronal PKA or related signaling molecules as potential therapeutic targets in gastrointestinal motility disorders.