We have determined the nucleotide sequence of the Dictyostelium discoideum 5.8S ribosomal RNA (rRNA). The sequence has relatively low homology with other 5.8S rRNAs and is further distinguished by its lack of modified nucleotides. The homology data indicate that D. discoideum diverged from the mainstream of eukaryotic descent at the earliest branch yet characterized by molecular phylogeny. Taking advantage of the considerable divergence of the D. discoideum 5.8S rRNA sequence from those of other eukaryotes, we have concluded that there is very little phylogenetically conserved, intramolecular secondary structure. This conclusion is discussed in the light of the variety of evidence for a highly ordered structure of 5.8S rRNA in vitro. We also offer comparative evidence in support of a specific model for the base pairing between the 3' end of 5.8S rRNA and the 5' end of eukaryotic 28S rRNA.