Formation of the dorsoventral axis in Drosophila melanogaster is mediated through control of the expression of several genes by the morphogen Dorsal. In the ventral part of the embryo Dorsal activates twist and represses zen amongst others. Recently, several proteins have been shown to assist Dorsal in the repression of zen, one of which is DSP1, a HMG box protein that was isolated as a putative co-repressor of Dorsal. In this report we used a DSP1 null mutant to ascertain in vivo the involvement of DSP1 in Dorsal-mediated repression of zen but not in the activation of twist. We show that Dorsal has the ability to interact with DSP1 in vitro as well as with rat HMG1. Using truncated versions of the proteins we located the domains of interaction as being the HMG boxes for DSP1 and HMG1 and the Rel domain for Dorsal. Finally, studies of the zen DNA binding properties of Dorsal and another related Rel protein (Gambif1 from Anopheles gambiae) revealed that their DNA binding affinities were increased in the presence of DSP1 and HMG1.