P2 is the major promoter for the murine c-myc proto-oncogene. The cis-acting elements that are required for initiation of transcription from P2 have not been well defined. In this report elements involved in initiating transcription from P2 were analysed in an in vitro system. In addition to a consensus TATA element at position -28, a guanine-rich element exists at position -48. This element, termed ME1a1, increases transcription initiation when inserted into a deletion mutant that lacks it. When mutations are engineered into ME1a1 it no longer acts to increase the level of initiation. Gel-shift and DNAase I footprinting analysis indicate that Me1a1 binds a protein. ME1a1 does not show any striking similarity to other promoter elements and therefore may be a novel cis-acting element.