Abstract Background: The Drosophila gene runt plays multiple roles during embryogenesis, including one as a pair‐rule class segmentation gene. The runt protein (Runt) contains an evolutionarily conserved domain (the Runt domain) that is found in several mammalian proteins including the human protein AML1, which is involved in many chromosome translocations associated with leukaemia. Specific DNA binding activity of a mammalian Runt domain is enhanced by a partner protein called PEBP2β/CBFβ. DNA binding activity of Drosophila Runt is also stimulated by this protein, suggesting the existence of a similar Runt partner protein in Drosophila. Results: We report here the cloning of two closely linked Drosophila genes, runt domain partner (rp) β1 and β2, that encode homologues of mouse PEBP2β/CBFβ. They are highly homologous to each other and to the mammalian counterpart. Either of the rpb proteins is capable of forming a complex with Runt and stimulating its DNA binding activity, but their temporal and spatial distributions are quite dissimilar, suggesting that functional specificity of Runt may be conferred by the interacting partner. Runt represses transcription dominantly when coexpressed with either partner in cultured cells, a function consistent with a direct role for Runt in regulating expression of the even‐skipped gene in Drosophila embryos. Conclusions: Drosophila Runt can interact with either of two Runt domain partners, and the resulting complex functions as an active repressor of transcription.