In the immunohistochemical staining of nerve growth factor, it has been reported that fixation-dependent lability of nerve growth factor hampers its localization. In the present study, we used two different polyclonal antibodies to immunostain nerve growth factor in rat brain tissue. We found that in paraformaldehyde-fixed (immersion- or perfusion-fixed) brains, nerve growth factor-like immunoreactivity was located primarily in the cytoplasmic membrane and fiber tract of hippocampal neurons and was sparse in cortical neurons. When fresh frozen brain sections were fixed in paraformaldehyde solution, nerve-growth factor-like immunoreactivity was distributed evenly in the cell body. However, when fresh frozen brain sections were fixed in acetone, immunoreactivity to nerve growth factor was present as discrete or confluent dense particles in the cell body, especially in the nuclear region. Also, when paraformaldehyde-perfusion-fixed brain sections were heat treated in salt solution before immunostaining, nerve growth factor-like immunoreactivity could be retrieved in the cytoplasmic and nuclear regions. The hippocampal formation, cerebral cortex and basal forebrain expressed nerve growth factor-like immunoreactivity. Double immunostaining in fresh frozen brains showed that the low-affinity nerve growth factor receptor (p75) co-expressed with nerve growth factor and trkA proto-oncogene in basal forebrain neurons. Our study shows that formaldehyde fixation can mask nerve growth factor antigen, and special treatment, such as heating, is needed to retrieve nerve growth factor antigen to permit immunohistochemical detection. For immunohistochemical study of nerve growth factor in rat brain tissue, successful immunostaining can be obtained by using fresh frozen brains to prevent the masking effect of fixatives or by using paraformaldehyde-fixed brains with heat treatment. It is likely that nerve growth factor is synthesized and accumulated mainly in the cell body but not in the fiber tracts, which is similar to the distribution of its messenger RNA. The co-existence of p75 with nerve growth factor and trkA in basal forebrain neurons suggests the role of low- and high-affinity receptors in regulating the trophic effect of nerve growth factor.