In this work, we have studied the role of the arginine finger region in determining the specificity of the GTPase activating proteins (GAPs) Saccharomyces cerevisiae Ira2p and human p120-GAP toward yeast Ras2p and human Ha-Ras p21. It is known that p120-GAP can enhance both Ras2p and Ha-Ras GTPase activities, whereas Ira2p is strictly specific for Ras2p and fails to activate Ha-Ras GTPase. Substitution in Ira2p of the arginine following the arginine finger with alanine, the residue found in the corresponding position of p120-GAP, or by glycine as found in neurofibromin, evokes a low but significant stimulation of Ha-Ras GTPase. The stimulatory activity of Ira2p on Ha-Ras increased by substituting segments of the finger loop region with p120-GAP residues, especially with the six residues forming the tip of the arginine loop. In p120-GAP, substitution of the entire finger loop with the corresponding region of Ira2p led to a construct completely inactive on Ha-Ras GTPase but active on yeast Ras2p GTPase. Analysis of these results and modeling of Ira2p. Ras complexes emphasize the importance of the finger loop region not only for the catalytic activity but also as a structural determinant involved in the specificity of GAPs toward Ras proteins from different organisms. Chemicals / CAS alanine, 56-41-7, 6898-94-8; arginine, 1119-34-2, 15595-35-4, 7004-12-8, 74-79-3; glycine, 56-40-6, 6000-43-7, 6000-44-8; guanosine triphosphatase, 9059-32-9; protein p21, 85306-28-1