The ecmA gene is specifically expressed in prestalk cells and its transcription is induced by the chlorinated hexaphenone DIF-1. We have purified a novel bZIP transcription factor, DimB, by affinity chromatography on two spatially separated ecmA promoter fragments. Mutagenesis of the cap-site proximal DimB-binding site (the -510 site) greatly decreases ecmA expression in the pstO cells, which comprise the rear half of the prestalk zone, and also in the Anterior-Like Cells, which lie scattered throughout the prespore region. However, DimB is not essential for normal expression of the ecmA gene, instead it spatially limits its expression; ecmA is relatively highly expressed in the subset of prestalk cells that coats the prestalk zone, but in slugs of a DimB-null strain, ecmA is highly expressed throughout the prestalk zone. Because the -510 site is required for correct ecmA expression, we posit a separate activator protein that competes with DimB for binding to the -510 site. DimB rapidly accumulates in the nucleus when cells are exposed to DIF-1, and ChIP analysis shows that, in the presence of extracellular cAMP, DIF-1 causes DimB to associate with the ecmA promoter in vivo. Thus, DIF-1 regulates DimB activity to generate a gradient of ecmA expression in the prestalk zone of the slug.