U8 snoRNA is the RNA component of a small nucleolar ribonucleoprotein (U8 snoRNP) required for accumulation of mature 5.8S and 28S rRNAs, components of the large ribosomal subunit. We have identified two putative U8 genes in Xenopus laevis. Sequence analysis of the coding regions of these two genes indicate that both differ at several positions from the previously characterized U8 RNA and that the two differ from each other. Functional analysis of these genes indicates that both are transcribed in vivo, produce stable U8 transcripts, and are capable of facilitating pre-rRNA processing in vivo. These data demonstrate that natural sequence variation exists among the U8 snoRNA genes in Xenopus. Alignment of these three Xenopus U8 sequences with the previously described mammalian U8 homologues in mouse, rat and human has provided information about evolutionarily conserved sequence and structural elements in U8 RNA. Identification and functional characterization of these naturally occurring variants in Xenopus has helped identify regions in U8 RNA that may be critical for function.