In H4IIE rat hepatoma cells insulin interacts with its receptors to induce DNA synthesis and promote cell division. However, the postreceptor events that lead to DNA synthesis and cell division have not been well characterized. Previous studies indicate that insulin can regulate the expression of several genes in H4 cells. One of these genes is the proto-oncogene c-fos, a cellular gene whose deregulation has been implicated in the process of cellular differentiation and division. In the present work insulin is shown to regulate cellular c-fos mRNA accumulation and the transcription rate of the c-fos gene. Insulin caused a rapid, dose-dependent increase in the cytoplasmic concentration of c-fos mRNA which was maximal by 30 min. Preceding this, a more rapid 6-8 fold increase in transcription of the c-fos gene was observed. Induction of transcription was apparent following only 5 min of insulin addition. This is the most rapid effect of insulin yet demonstrated on the induction of gene expression. Protein synthesis inhibitors (cycloheximide, anisomycin) also induced the transcription of the c-fos gene. However, they stimulated a much greater increase in transcription than did insulin, and followed a different time course of action. The addition of insulin in combination with a protein synthesis inhibitor resulted in no greater increase in c-fos transcription than did the addition of a protein synthesis inhibitor alone. The nonadditivity of H4 cell c-fos gene expression may indicate a similar mode of action by insulin and protein synthesis inhibitors.