The runx gene family encodes transcriptional regulators that are essential in several processes. Alternative transcription start sites (3' or P1 and 5' or P2) have been described both at runx1 and runx2. Functional domains have been mapped to P2 N-termini. Here, starting from an analysis of cDNA clones, we identify a P2 promoter at mouse runx3. In functional assays in COS-7 cells, this promoter is as active as runx1 P2. By nuclease protection, primer extension and deletion analysis we map a major start site and define a minimal promoter element. The activity of both promoters at all three mouse loci was systematically compared in a comprehensive panel of fresh tissues. The conservation of a two-promoter structure and their distinctive pattern of activity support the hypothesis that alternative 5' isoforms subserve unique functions.