The bovine complementary DNA encoding the catalytic domain of Ras GTPase activating protein was mutagenized semirandomly using a variation of the polymerase chain reaction. Sixty-four mutated codons were identified with seventeen of the mutations deleterious to Ras GTPase activating function. All of the inactivating single mutations affected the structure of the catalytic fragment as assessed by large decreases in soluble protein when expressed in Escherichia coli. Upon examination of the Ras binding properties of 10 of the mutants, only 1 was measurably impaired for Ras binding and 4 appeared to have increased affinity for Ras. These results demonstrate that Ras binding and GTPase activation are two distinct properties of GTPase activating protein. Additionally, the catalytic mechanism of GTPase activating protein is much more sensitive to structural perturbation than is Ras binding.