The synapse is the primary locus of cell-cell communication in the nervous system. It is now clear that the synapse incorporates diverse cell signaling modalities in addition to classical neurotransmission. Here we show that two components of the insulin pathway are localized at CNS synapses, where they are components of the postsynaptic density (PSD). An immunochemical screen revealed that polypeptides of 58 and 53 kDa (p58/53) were highly enriched in PSD fractions from rat cerebral cortex, hippocampus, and cerebellum. These polypeptides were purified and microsequenced, revealing that p58/53 is identical to the insulin receptor tyrosine kinase substrate p58/53 (IRSp53). Our analysis of IRSp58/53 mRNA suggests that within rat brain there is one coding region for IRSp58 and IRSp53; we find no evidence of alternative splicing. We demonstrate that IRSp58/53 is expressed in the synapse-rich molecular layer of the cerebellum and is highly concentrated at the synapses of cultured hippocampal neurons, where it co-localizes with the insulin receptor. Together, these data suggest that insulin signaling may play a role at CNS synapses.