Insulin stimulates RNA synthesis in the human hepatoma cell line J5. The mechanisms of insulin action on nuclear functions are not fully understood. To assess the possible action of nuclear insulin binding on RNA synthesis, we studied the insulin action by an in vitro transcription system using the nuclei isolated from control cells and insulin-stimulated J5 cells. Insulin binding was done by ligand binding assay. RNA synthesis was also studied in the nuclei treated with insulin in vitro. We found that a large pool of low-affinity binding sites existed in the isolated nuclei preparation without site-to-site interactions. These data suggest that the majority of nuclear insulin binding sites are located inside the nuclear envelope. Nuclear RNA synthesis was stimulated by the insulin (about a 4-fold increase when compared to the control) added to intact cells but not to isolated nuclei. We conclude that insulin stimulates nuclear RNA synthesis via interaction with plasma membrane receptors.