The c-myc promoter has the unusual property of displaying both RNA polymerase II (Pol II) and RNA polymerase III (Pol III) activities. Both Pol II and Pol III utilize the same transcription initiation site. We have now examined the effects of mutations in crucial regions of the c-myc promoter to assess their effects on both transcriptional activities. In doing this we show that both Pol II and Pol III activities require sequences that are located within the stronger of the two principal c-myc promoter regions (P2). Further, we show that the Pol III activity using this initiation site does not require an A box or distal upstream sequences. Like the Pol II activity, it does require an intact TATA sequence and alterations at this site result in the simultaneous loss of both Pol II and Pol III activities. The superimposition of two apparently inseparable promoter activities makes it possible to consider common features, possible common protein elements in each holoenzyme complex, as well as a potential role for each enzyme in the regulated expression of the c-myc gene.