Vertebrate genomes contain proto-oncogenes whose enhanced expression or alteration by mutation seems to be involved in the development of naturally occurring tumours. These activated genes, usually assayed by their ability to induce the malignant transformation of NIH 3T3 cells, are frequently related to the ras oncogene of Harvey (Ha-ras) or Kirsten (Ki-ras) murine sarcoma viruses, or a third member of this family (N-ras). Activation involves point mutation which often affect codon 12 (refs 16-26) of the encoded 21,000-molecular weight polypeptide (p21). To provide insight into structural requirements involved in p21 activation, we have now constructed 20 mutant c-Ha-ras1 genes by in vitro mutagenesis, each encoding a different amino acid at codon 12. Analysis of rat fibroblasts transfected with these altered genes demonstrates that all amino acids except glycine (which is encoded by normal cellular ras genes) and proline at position 12 activate p21, suggesting a requirement for an alpha-helical structure in this region of the polypeptide. The morphological phenotype of cells transformed by the activated genes can, however, depend on the particular amino acid at this position.