Immunocytochemical studies have been performed which establish the presence of immunoreactive (ir-) insulin in primary cultures from fetal rat brain. Approximately 1% of the identified neurons in culture contained insulin immunoreactivity. Typically, a diffuse staining pattern was observed in the perikaryia and fiber profiles of peroxidase-anti-peroxidase-positive neurons. Varicosity-like structures containing ir-insulin were found on the majority of positively stained fiber profiles. A small number of the neurons appeared to have cytoplasmic granules that stained densely for insulin. No significant staining was observed in the background monolayer of cells predominantly of glial origin. The substitution of primary antiserum absorbed with excess insulin or preimmune serum eliminated staining in the neurons. The observation of insulin immunoreactivity in neurons suggests that insulin may be a neuromodulator in the central nervous system.