The rnal-1 mutation of Saccharomyces cerevisiae is a recessive and temperature sensitive lethal mutation which interferes with mRNA, rRNA and tRNA production. The complete nucleotide sequence of the RNAI gene of S. cerevisiae was determined by dideoxy chain termination method. RNAI gene, composed of a 1221 bp open reading frame, encodes a 407 amino acid-protein. TATA box, CAAT box and polyadenylations signal (AATAAA) are found, but CT block and pyAAG sequence reported to enhance transcriptional efficiency are not found. RNAI gene protein contains many acidic amino acids such as GLU and ASP in the C-terminus region. A recombinant plasmid was constructed to overproduce the RNAI gene product using phage lambda P_L promoter.