The experiments reported here used 3T6-Swiss albino mouse fibroblasts and H4-II-E-C3 rat hepatoma cells as model systems to examine the mechanism(s) through which insulin regulates rDNA transcription. Serum starvation of 3T6 cells for 72 h resulted in a marked reduction in rDNA transcription. Treatment of serum-deprived cells with insulin was sufficient to restore rDNA transcription to control values. In addition, treatment of exponentially growing H4-II-E-C3 with insulin stimulated rDNA transcription. However, for both cell types, the stimulation of rDNA transcription in response to insulin was not associated with a change in the cellular content of RNA polymerase I. Thus we conclude that insulin must cause alterations in formation of the active RNA polymerase I initiation complex and/or the activities of auxiliary rDNA transcription factors. In support of this conclusion, insulin treatment of both cell types was found to increase the nuclear content of upstream binding factor (UBF) and RNA polymerase I-associated factor 53. Both of these factors are thought to be involved in recruitment of RNA polymerase I to the rDNA promoter. Nuclear run-on experiments demonstrated that the increase in cellular content of UBF was due to elevated transcription of the UBF gene. In addition, overexpression of UBF was sufficient to directly stimulate rDNA transcription from a reporter construct. The results demonstrate that insulin is capable of stimulating rDNA transcription in both 3T6 and H4-II-E-C3 cells, at least in part by increasing the cellular content of components required for assembly of RNA polymerase I into an active complex.