The effect of electrolytic lesions of the neonatal forebrain on the morphogenesis of the mouse neocortex has been examined. Balb/C mice were lesioned unilaterally within 24 h of birth. The development of cortical cytoarchitecture was assessed in Nissl-stained sections, and the levels of presynaptic markers for cholinergic, noradrenergic and serotonergic afferents were measured in the fronto-parietal cortex ipsilateral and contralateral to the lesion at various postnatal ages and in adulthood. The basal forebrain (nBM) lesion resulted in a transient but severe reduction of cortical cholinergic markers and in abnormal cortical cytoarchitecture. Cytoarchitectural abnormalities were expressed as delay in the emergence of differentiated cell populations and affected sequentially more superficial layers with maturation following lesion. Furthermore, the location and extent of these morphologic abnormalities appeared to correlate with the degree of cholinergic denervation. Cortical monoamines were also temporarily reduced as a result of the lesion; however, pharmacologic lesions of the monoaminergic projections alone did not result in the abnormal cortical cytoarchitecture. Thus, the basal forebrain cholinergic projection appears to serve a role in regulating cortical differentiation.