The specific and dynamic RNA:RNA interactions between pre-mRNA and small nuclear RNAs (snRNAs), especially U2, U5, and U6 snRNAs, form the catalytic core and are at the heart of the spliceosome formation. The functionally important regions in the snRNAs correspond to the highly modified regions in snRNAs from human, rat, and plant cells. To better understand the importance of the modifications of snRNAs, we identified and localized the modified nucleotides in the five spliceosomal snRNAs of Schizosaccharomyces pombe cells. Twenty-two modified nucleotides, including base methylations, 2'-O-methylations, and pseudouridines, were found in the five spliceosomal snRNAs. The conservation of modified nucleotides between human and S. pombe snRNAs is striking. In addition, most of the modified nucleotides are in or around positions that form hydrogen bonds with the pre-mRNA or with other snRNAs. The results are consistent with the suggestion that modified nucleotides are clustered around functionally important regions of the spliceosomal snRNAs. These data provide the basis for further functional studies on posttranscriptional modifications in spliceosomal snRNAs.