Polypeptide chain elongation factor 1 alpha (EF-1 alpha) of Saccharomyces cerevisiae is encoded by two distinct genes designated EF1 alpha A and EF1 alpha B [Nagata et al., EMBO J. 3 (1984) 1825-1830]. Both genes were cloned, and their nucleotide (nt) sequences were determined [see also Schirmaier and Philippsen, EMBO J. 3 (1984) 3311-3315, and Cottrelle et al., J. Biol. Chem. 260 (1985) 3090-3096]. They contain an open reading frame of 1374 nt coding for an identical protein of 458 amino acid residues, although their nt sequences differed at two positions. In this paper, we determined their 5'- and 3'-flanking sequences which were considerably different each other. From the S1 nuclease mapping of mRNA, both genes are found to be expressed almost to the same extent in exponentially growing cells. The transcription start points for EF1 alpha A and EF1 alpha B mRNAs were precisely located by primer extension procedure at 32 and 23 nt upstream of the start codons, respectively. The sequence which commonly exists in the 5'-flanking regions of ribosomal protein genes of S. cerevisiae was also present in the two EF1 alpha genes.