Basal forebrain cholinergic neurons (BFCNs) are involved in cognitive functions such as learning and memory, and are affected in several neurodegenerative diseases (e.g. Alzheimer's disease). Despite their importance, the molecular mechanisms of their development are not fully elucidated. A recent report demonstrated that some BFCNs in adult rat are positive for L3/Lhx8, a LIM homeobox transcription factor. To examine the function of L3/Lhx8 in the development of BFCNs, we generated L3/Lhx8 gene-disrupted mice. In these mice, cells expressing cholinergic neuron markers, such as choline acetyltransferase, vesicular acetylcholine transporter and p75 low-affinity NGF receptor, were markedly reduced in the basal forebrain, whereas other cholinergic neurons including brain stem and spinal motor neurons expressed the markers. Neurotransmitter phenotypes other than cholinergic in the basal forebrain appeared intact. From these results, we suggested that L3/Lhx8 has a pivotal and specific role in the development and/or maintenance of BFCNs.