In this study, we show that the Saccharomyces cerevisiae ORF YBR142w, which encodes a putative DEAD-box RNA helicase, corresponds to MAK5. The mak5-1 allele is deficient in the maintenance of the M1 dsRNA virus, resulting in a killer minus phenotype. This allele carries two mutations, G218D in the conserved ATPase A-motif and P618S in a non-conserved region. We have separated these mutations and shown that it is the G218D mutation that is responsible for the killer minus phenotype. Mak5p is an essential nucleolar protein; depletion of the protein leads to a reduction in the level of 60S ribosomal subunits, the appearance of half-mer polysomes, and a delay in production of the mature 25S and 5.8S rRNAs. Thus, Mak5p is involved in the biogenesis of 60S ribosomal subunits.