Small nucleolar RNAs (snoRNAs) play an important role in eukaryotic rRNA biogenesis. By combination of a computer search of EMBL database and experimental procedure, a novel snoRNA coding sequence (Z8) was screened out and characterized from yeastSaccharomyces cerevisiue genome. Z8 snoRNA gene codes a boxC/D antisense snoRNA which guides, deduced from structure analysis, the 2'-O-ribose methylation at U(2421) of 25S rRNA. After disruption of Z8 snoRNA gene, the methylation at corresponding site was abolished, but no gmwth delay was observed in various cultural temperatures. Z8 DNA is the first gene of a gene cluster consisting of three cognate snoRNA genes which are located on an intergenic region of chromosome XIII. This gene cluster is co-transcribed as a polycistronic precursor from a + 247 bp U snoRNA gene promoter, followed by processing to release individual snoRNAs, representing a new expression pattern of snoRNA genes.