Three novel small noncoding RNAs were identified from the conserved intronic regions of D. melanogaster by using comparative genomics method and molecular biology approach. One is a novel snoRNA in a combinated way, which displays structural features typical of C/D box snoRNA family and possesses a 10-nt-long rRNA antisense element for guiding the 2'-O-methylation of the D. melanogaster 28S rRNA at C2673. The other two are miRNAs whose predicted precursors adopt a stem-loop structure characteristic of known miRNA. The two miRNAs genes appear to have ubiquitous expression profiles with similar to 23-nt RNA transcripts detected by Northern blotting. Our study revealed 396 multi-species intronic conserved sequences (MCIS) that nested in the introns with a length from 100 to 500 bp. In addition to small RNA coding, the MCIS might function as cis-acting elements involved in gene transcription or post-transcriptional processing.