Abstract: We have cloned a single copy gene that encodes a small nuclear RNA, designated snR3, from the yeast Saccharomyces cerevisiae. This RNA is highly conserved among fungi, and sequence and secondary structure analyses suggest that snR3 is analogous to mammalian U4 snRNA. To determine whether snR3 has an essential function in yeast, the gene (designated SNR3), was disrupted by replacing 35 nucleotides of coding sequences with 2.2 kb of yeast DNA containing the LEU2 gene. Since cells entirely lacking snR3 were expected to be inviable, the nonfunctional gene was used to replace one chromosomal copy in a diploid cell, and the diploid transformants were sporulated. Surprisingly, virtually all tetrads gave rise to four viable spores. Moreover, these haploid strains, which have been shown by DNA blot hybridization to lack an intact copy of the SNR3 gene, and which contain no detectable snR3 transcripts, are indistinguishable from their SNR3+ sister spores under a variety of growth conditions.