The neurotransmitter acetylcholine is an important modulator of cognitive functions including attention, learning, and memory. The actions of acetylcholine are mediated by five distinct muscarinic acetylcholine receptor subtypes (M(1)-M(5)). The lack of drugs with a high degree of selectivity for these subtypes has impeded the determination of which subtypes mediate which components of cholinergic neurotransmission relevant to cognitive abilities. The present study examined the behavioral functions of the M(2) muscarinic receptor subtype by utilizing congenic C57BL/6 mice possessing a null-mutation in the M(2) muscarinic receptor gene (M(2)(-/-) mice). Comprehensive assessment of general health and the neurological function found no major differences between M(2)(-/-) and wild-type (M(2)(+/+)) mice. In the tests of learning and memory, M(2)(-/-) mice were impaired in the acquisition (trials to criterion), but not the retention (72h) of a passive avoidance task. In a novel open field, M(2)(-/-) mice were impaired in between-sessions, but not within-session habituation. In a holeboard test of spatial memory, M(2)(-/-) mice committed more errors in working memory than M(2)(+/+) mice. Reference memory did not differ between the genotypes. M(2)(-/-) mice showed no impairments in either cued or contextual fear conditioning. These findings replicate and extend earlier findings in a hybrid strain and solidify the interpretation that the M(2) receptor plays a critical role in specific components of cognitive abilities.