Abstract: The RNA helicase gene family encodes a group of eight homologous proteins that share regions of sequence similarity. This group of evolutionarily conserved proteins presumably all utilize ATP (or some other nucleoside triphosphate) as an energy source for unwinding double-stranded RNA. Members of this family have been implicated in a variety of physiological functions in organisms ranging from Escherichia coli to human, such as translation initiation, mitochondrial mRNA splicing, ribosomal assembly, and germinal line cell differentiation. We have applied polymerase chain reaction technology to search for additional members of the RNA helicase family in the yeast Saccharomyces cerevisiae. Using degenerate oligonucleotide primers designed to amplify DNA fragments flanked by the highly conserved motifs V L D E A D and Y I H R I G, we have detected five putative RNA helicase genes. Northern and Southern blot analyses demonstrated that these genes are single copy and expressed in yeast. Several members of the RNA helicase family share sequence identity ranging from 49.2% to 67.2%, suggesting that they are functionally related. The discovery of such a multitude of putative RNA helicase genes in yeast suggests that RNA helicase activities are involved in a variety of fundamentally important biological processes.