Nucleotide sequences proximal to the initiation codon of a gene are known to affect the expression efficiency of that gene. We screened 10-bp random sequences upstream of the initiation codon of the zeocin-resistance gene to identify sequences that could enhance its expression in Saccharomyces cerevisiae. Of the isolated sequences, 20 sequences exhibited a common feature, i.e. ATG at the position -9 through -7, which resulted in the incorporation of three amino acids at the N-terminus of the protein. The introduction of these sequences upstream of the initiation codon increased the expression levels of zeocin-resistance protein by 2.2-6.5-fold. One of these sequences increased the expression levels of three out of four human proteins, thereby suggesting that this sequence may also enhance the expression efficiency of mammalian proteins in yeast.