This study demonstrates that a deficiency of forebrain cholinergic neurons occurs in C57BL/6 (C57) mice, a strain characterized by poor learning capabilities. The brains of 21-day-old and 18-week-old C57 and DBA/2 (DBA) mice were studied by means of acetylcholinesterase (AChE) histochemistry and of choline acetyltransferase (ChAT) immunocytochemistry. Computer-assisted image analysis was performed on sections through the medial septum, the diagonal band of Broca, the basal nucleus of Meynert and the neostriatum. As compared to the DBA strain, C57 mice had a reduced number of forebrain cholinergic neurons. This feature was present at the age of 21 days and persisted to 18 weeks. Between-strain variations in the density of neurons were more obvious in ChAT-stained material than in AChE-stained sections. These data show that C57 mice can be regarded as a genetic mutant, whose phenotype is characterized by a reduced number of forebrain cholinergic neurons and by cognitive abnormalities. C57 mice represent a valuable model for studying the influence of genetic factors on central nervous system cholinergic mechanisms and the effects of genetically determined cholinergic deficiency on behavior and learning.