TC21 is the fourth member of the ras gene family to exhibit oncogenic activation in human tumor cells. To assess the prevalence of activated TC21 oncogenes in human tumors, we have developed sensitive single-strand conformational polymorphism (SSCP) conditions and immunological reagents for the detection of both single base alterations and/or overt overexpression in a wide spectrum of human tumor cell lines and surgical samples. In an initial examination of 33 human tumor specimens, we observed a novel nine basepair three amino acids insertion at TC21 codon 24 in one human uterine leiomyosarcoma cell line, SK-UT-1. This mutant allele when transfected into NIH3T3 cells, displayed high transforming activity comparable to that of the Leu72 oncogenic mutant identified by expression cDNA cloning from a human ovarian carcinoma cell line. Comparing the level of GTP-binding by the mutant and normal TC21 products revealed that this novel lesion increases the GTP-bound form of the TC21 molecule. These findings imply that the mechanism by which mutations activate the oncogenic properties of this ras-related molecule is analogous to that of previously known ras family members.