Using the yeast two-hybrid system, we have identified developmentally regulated Dictyostelium genes whose encoded proteins interact with Ras-GTP but not Ras-GDP. By sequence homology and biochemical function, one of these genes encodes a Ras GAP (DdRasGAP1). Cells carrying a DdRasGAP1 gene disruption (ddrasgap1 null cells) have multiple, very distinct growth and developmental defects as elucidated by examining the phenotypes of ddrasgap1 null strains. First, vegetative ddrasgap1 null cells are very large and highly multinucleate cells when grown in suspension, indicating a severe defect in cytokinesis. When suspension-grown cells are plated in growth medium on plastic where they attach and can move, the cells rapidly become mono- and dinucleate by traction-mediated cell fission and continue to grow vegetatively with a number of nuclei (1-2) per cell, similar to wild-type cells. The multinucleate phenotype, combined with results indicating that constitutive expression of activated Ras does not yield highly multinucleate cells and data on Ras null mutants, suggest that Ras may need to cycle between GTP- and GDP-bound states for proper cytokinesis. After starvation, the large null cells undergo rapid fission when they start to move at the onset of aggregation, producing mononucleate cells that form a normal aggregate. Second, ddrasgap1 null cells also have multiple developmental phenotypes that indicate an essential role of DdRasGAP1 in controlling cell patterning. Multicellular development is normal through the mid-slug stage, after which morphological differentiation is very abnormal and no culminant is formed: no stalk cells and very few spores are detected. lacZ reporter studies show that by the mid-finger stage, much of the normal cell-type patterning is lost, indicating that proper DdRasGAP1 function and possibly normal Ras activity are necessary to maintain spatial organization and for induction of prestalk to stalk and prespore to spore cell differentiation. The inability of ddrasgap1 null cells to initiate terminal differentiation and form stalk cells is consistent with a model in which Ras functions as a mediator of inhibitory signals in cell-type differentiation at this stage. Third, DdRasGAP1 and cAMP dependent protein kinase (PKA) interact to control spatial organization within the organism. Overexpression of the PKA catalytic subunit in ddrasgap1 cells yields terminal structures that are multiply branched but lack spores. This suggests that RasGAP and PKA may mediate common pathways that regulate apical tip differentiation and organizer function, which in turn control spatial organization during multicellular development. It also suggests that DdRasGAP1 either lies downstream from PKA in the prespore to spore pathway or in a parallel pathway that is also essential for spore differentiation. Our results indicate that DdRasGAP1 plays an essential role in controlling multiple, potentially novel pathways regulating growth and differentiation in Dictyostelium and suggest a role for Ras in these processes.