Haploid cells of budding yeast Saccharomyces cerevisiae respond to mating pheromones by inducing genes required for conjugation, arresting cell cycle progression, and undergoing morphological changes. The same cells respond to nutrient deprivation by altering budding pattern and inducing genes required for invasive growth. Both developmental alternatives to vegetative proliferation require the MAP kinase Kss1 and the transcriptional transactivator Ste12. Using a two-hybrid screen for gene products that interact with Kss1, two homologous and previously uncharacterized loci (DIG1 and DIG2, for down-regulator of invasive growth) were identified. DIG2 is pheromone-inducible, whereas DIG1 is constitutively expressed. Dig1 colocalizes with Kssl in the nucleus, coimmunoprecipitates with Kss1 from cell extracts in a pheromone-independent manner, and is phosphorylated by Kss1 in immune complexes in a pheromone-stimulated manner. Kss1 binds specifically to a GST-Dig1 fusion in the absence of any other yeast protein. Using the two-hybrid method, both Dig1 and Dig2 also interact with the other MAP kinase of the pheromone response pathway, Fus3. However, neither dig1 or dig2 single mutants, nor a dig1 dig2 double mutant, have a discernible effect on mating. In contrast, dig1 dig2 cells constitutively invade agar medium, whereas a dig1 dig2 ste12 triple mutant does not, indicating that Dig1 and Dig2 share a role in negatively regulating the invasive growth pathway. High-level expression of Dig1 suppresses invasive growth and also causes cells to appear more resistant to pheromone-imposed cell cycle arrest. Ste12 also binds specifically to GST-Dig1 in the absence of any other yeast protein. Collectively, these findings indicate that Dig1, and most likely Dig2, are physiological substrates of Kssl and suggest that they regulate Ste12 function by direct protein-protein interaction.