A rat beta-nerve growth factor (NGF) genomic sequence encoding the entire 3' exon of preproNGF was cloned, and its nucleotide sequence was determined. Rat NGF shows very high homology with other known NGFs in both the prepropeptide and the 3' untranslated regions. The presumptive signal sequence, the cysteine residues important for tertiary structure, possible glycosylation sites, and dibasic amino acids required for proteolytic cleavage to mature NGF are conserved across species. Comparison of the hydrophobicity plots and amino acid sequences revealed an evolutionary divergent domain on the external surface of NGF, which may account for the poor immunologic crossreactivities of the various NGFs. In situ hybridization with a rat-specific oligodeoxynucleotide indicated high levels of NGF mRNA synthesis in both hippocampal granule and pyramidal cell layers. These results are consistent with one role for NGF in the CNS as a neuronally released, retrogradely transported neurotrophic factor for basal forebrain cholinergic neurons.