A-raf gene was first detected by low-stringent hybridization with v-raf and was reported as a possible oncogene. We have cloned rat A-raf cDNA and determined the complete nucleotide sequence over its long open reading frame. Alignment of rat A-raf- and c-raf-deduced amino acid sequences shows 59.4% homology. Both the ATP binding site and the kinase domain are conserved in A-raf as in c-raf. Furthermore, three regions highly conserved between A-raf and c-raf were identified in the 5'-half region of c-raf which has been shown to be deleted in the activated form of c-raf. These regions were speculated to be the candidates of the regulatory domain controlling the protein kinase activity of raf products. Southern blot analysis shows that genes homologous to A-raf and c-raf are present as independent loci in Drosophila melanogaster, Caenorhabditis elegans, Saccharomyces cerevisiae and Candida boidinii. A-raf was expressed in several tissues and cell line at various amounts.