In peripheral insulin-sensitive tissues, insulin receptor substrate (IRS-1) undergoes tyrosine phosphorylation immediately after cells are stimulated by insulin or insulin-like growth factor-1 (IGF-1), and may function as a molecular link between insulin/IGF-1 receptor tyrosine kinases and enzymes regulating cell growth and metabolism. A fundamental question pertaining to insulin/IGF-1 action in the brain is whether IRS-1 is expressed by neurons. In this study, the distribution of cells containing immunoreactivity to IRS-1 in the brain was determined by immunocytochemistry with polyclonal IRS-1 antiserum, and compared to the localization of immunostaining for phosphotyrosine using polyclonal phosphotyrosine antiserum. The immunostaining results with ABC-peroxidase method and cryostat sections showed the presence of IRS-1 immunoreactivity in many neuron cell bodies throughout the rat forebrain, particularly in the habenula, cerebral cortex and piriform cortex. In the hypothalamus, IRS-1 immunostaining was present in neurons of the paraventricular nucleus, supraoptic nucleus, and arcuate nucleus. The choroid plexus stained intensely for IRS-1. The populations of cells that stained for IRS-1 also showed strong immunostaining for phosphotyrosine. Studies at the cellular level are needed to verify coexpression of IRS-1 and receptors for insulin or IGF-1 by the same neurons, as well as in cells of the choroid plexus. The present results are the first demonstration of IRS-1 expression by neurons in adult mammalian brain. These findings are consistent with the hypothesis that insulin and IGF-1 actions in the brain involve signal transduction mechanisms common to those found in peripheral tissues.