U21 is an intron-encoded snoRNA in vertebrates which contains a 13-nt tract of complementarity to an invariant sequence in eukaryotic 28S rRNA. Here, we report the characterization of its Drosophila melanogaster homolog which is the first case of an intron-encoded snoRNA in an invertebrate metazoan. In D. melanogaster, U21 is encoded within the ARF-1 (ADP ribosylation factor 1) gene, whereas in chicken and mammals it is found in the ribosomal protein L5 gene. In D. melanogaster, like in vertebrates, U21 is devoid of a 5' trimethylguanosine cap, thus, likely resulting from processing of intronic RNA. The only portion of U21 sequence preserved between D. melanogaster and vertebrates, in addition to the hallmark box C and box D motifs, corresponds precisely to the 13-nt complementary to rRNA, pointing to an important role of the pairing of U21 to pre-rRNA.