Nerve growth factor (NGF) injected into the otherwise unlesioned adult rat septum induced sprouting of presumptive cholinergic fibres positive for p75NGFR and acetylcholinesterase (AChE). These fibres did not stain for tyrosine hydroxylase and therefore did not represent sympathetic ingrowth. Neurofilament staining on adjacent sections revealed fibres with similar morphology, suggesting new outgrowth in the form of sprouting rather than the upregulation of p75NGFR and AChE in pre-existing fibres. Simultaneous injections of subneurotoxic doses of N-methyl-D-aspartate (NMDA) significantly potentiated the effect of NGF on cholinergic fibre sprouting and caused pronounced glial fibrillary acidic protein (GFAP)-positive astrocytosis. Application of NMDA alone did not elicit sprouting of this type. These findings indicate that NGF can induce the sprouting of uninjured adult rat septal cholinergic fibres in vivo and suggest that reactive astrocytes are not inhibitory to cholinergic axonal outgrowth, and might serve as a substrate for growing axons in the presence of NGF.