Insulin directly affects many aspects of cellular metabolism. An additional, poorly studied effect of insulin is the regulation of multiple genes whose products are important in many cellular functions. Using differential screening techniques, we identified insulin-regulated genes induced in insulin-treated rat H4IIE (H4) hepatoma cells. Two of the mRNAs identified were homologous to the previously characterized mouse pip 92 and rat CL-6 immediate-early genes. The pip 92 clone was initially isolated from mitogen-stimulated mouse Balb/c 3T3 fibroblasts, whereas the CL-6 clone was first isolated from regenerating rat liver. In this article, we demonstrated that in rat H4 cells, the transcription rates of both pip 92 and CL-6 are induced by insulin alone. Additionally, we showed that the transcription rates of two other genes, whose sequences are not homologous to any other sequences in gene bank ("novel genes"), were rapidly and transiently induced by insulin. These results demonstrate that insulin regulates the expression of several novel genes with a time-course similar to members of the immediate-early response gene family.