The small GTP-binding protein rac-1, a member of the ras gene superfamily of GTPases, is thought to be a key component of a signal transduction pathway that mediates cell membrane ruffling and actin stress fiber formation induced by growth factors. rac-1 protein is regulated by the interplay of several activities: proteins that enhance GDP dissociation (GDP Dissociation Stimulator, GDS), inhibit nucleotide exchange (GDP Dissociation Inhibitor, GDI), or accelerate GTP hydrolysis (GTPase Activating Protein, GAP). We have assessed the relative contribution of the rac-1/GAP interactions to the overall activity of rac-1 by expressing alpha 1-chimaerin, a rac-1-specific GAP, in fibroblasts. NIH 3T3 cells were transfected with alpha 1-chimaerin cDNA-containing expression vector and stable clones were established. Extracts prepared from alpha 1-chimaerin-expressing cells showed rac-1 GAP activity that was regulated by phosphatidylserine and phorbol ester. The cells expressing alpha 1-chimaerin showed a distinct phenotype. They had altered adhesive properties as measured by their ability to bind to a fibronectin-coated glass surface, suggesting that the expression of a rac-1 GAP alters the assembly of integrin receptors, actin and cytoskeletal proteins such as vinculin and talin. Direct demonstration of this phenomenon was achieved by studying the organization of actin stress fiber and formation of focal adhesions in the alpha 1-chimaerin expressing cells following stimulation by growth factors. Mock transfected cells, upon serum or lysophosphatidic acid stimulation, organize actin as a dense array of parallel fibers running the length of the cell. This process did not take place in the cells expressing rac-1 GAP. Similarly, the formation of focal adhesions as measured by the appearance of vinculin clusters was impaired in the alpha 1-chimaerin expressing cells. These results demonstrate that expression of a GAP for rac-1 in fibroblasts produces profound changes in the cytoskeletal organization and suggest that GAP activity negatively regulates rac-1 function.