The c-ets-1 locus encodes two transcription factors, p54c-ets-1 and p68c-ets-1 that recognize purine-rich motifs. The v-ets oncogene of the avian retrovirus E26 differs from its cellular progenitor p68c-ets-1 by two amino acid substitutions (alanine 285 and isoleucine 445 in c-ets-1 both substituted by valine in v-ets, mutations A and B respectively) and its carboxy-terminal end (mutation C). The B mutation affects a well conserved residue in the carboxy-terminal 85 amino acids, ETS DNA-binding domain. To address the biological relevance of the B mutation found between v-ets and c-ets-1, we have randomly mutagenized isoleucine 445 of p68c-ets-1 by polymerase chain reaction. Using in vitro gel mobility shift assays, we show that this residue is crucial for the binding properties of c-ets-1 since the 12 mutations we have generated at this position, all diminish or even abolish the binding, to the 'optimized' Ets-1 binding site (EBS), of 35 kDa proteins corresponding to the 311 carboxy-terminal residues of c-ets-1. Among them, substitutions of isoleucine to glutamic acid, glycine or proline have the highest inhibitory effects. Similar results were obtained when the same mutations were introduced either in full-length p68c-ets-1 protein or into a carboxy-terminal polypeptide of 109 amino acids encompassing the ETS-domain which has previously been shown to display a very high binding activity as compared with the full-length protein. Consistent with the in vitro results, point mutations in p68c-ets-1 that decrease binding activity to EBS abrogate its ability to transactivate reporter plasmids carrying either the TPA Oncogene Response Unit of the Polyoma virus enhancer (TORU) or a sequence derived from the HTLV-1 LTR. Furthermore, as this isoleucine residue is rather well-conserved within the ETS gene family, we show that mutation of the corresponding isoleucine of c-ets-2 into glycine also abrogates its DNA-binding and hence, transactivating properties. Thus, the v-ets B mutation highlights the isoleucine 445 as an essential amino acid of the c-ets-1 and c-ets-2 DNA-binding domains.