Transcripts for the β2 and the β4 nicotinic acetylcholine receptor (nAChR) subunits are found throughout the CNS and the peripheral nervous system. These two β subunits can form heteromultimeric channels with any of the α2, α3, α4, or α5 subunits in heterologous expression systems. Nonetheless, the subunit composition of native nAChRs and the role of different nAChR subtypesin vivo remain unclear. We prepared null mutations for the β2 and the β4 genes and bred β2−/−β4−/− mice by mating mice of identical β2−/−β4+/− or β2+/−β4−/− genotype. The β2−/− and the β4−/− single-mutant mice grow to adulthood with no visible phenotypic abnormalities. The β2−/−β4−/− double mutants survive to birth but have impaired growth and increased perinatal mortality. They also present enlarged bladders with dribbling urination and develop urinary infection and bladder stones. The ocular pupils are widely dilated and do not constrict in response to light. Histological studies revealed no significant abnormalities of brain or peripheral tissues except for hyperplasia in the bladder mucosa of β4−/− and β2−/−β4−/− mutants. Bladder strips from β2−/−β4−/− mice did not respond to nicotine but contracted when stimulated with a muscarinic agonist or electric field stimulation. Bladder strips from β4 mutants did not respond to nicotine despite the absence of major bladder dysfunction in vivo. Acetylcholine-activated whole-cell currents were absent in superior cervical ganglion neurons from β2−/−β4−/− mice and reduced in neurons from β4−/− mice. Although there is apparent redundancy and a superficially normal phenotype in β2−/− and β4−/− mice, physiological studies indicate major deficits in the β4−/− mice. Our previous description of a similar phenotype in α3−/− mice and the current data suggest that the α3 and the β4 subunits are major components in autonomic nAChRs. The phenotype of the β2−/−β4−/− and α3−/− mice resembles the autosomal recessive megacystis-microcolon-hypoperistalsis syndrome in humans.