Cytoplasmic ribosomes from Euglena gracilis contain 16 rRNA components. These include the typical 5 S, 5.8 S and 19 S rRNAs that are found in other eukaryotes as well as 13 discrete small RNAs that interact to form the equivalent of eukaryotic 25-28 S rRNA (accompanying paper). We have utilized DNA sequencing techniques to establish that genes for all of these RNAs, with the exception of 5 S rRNA, are encoded by the 11,500 base-pair circular rDNA of E. gracilis. We have determined the relative positions of the coding regions for the 19 S rRNA and the 14 components (including 5.8 S rRNA) of the large subunit rRNA, thereby establishing that the genes for each of these rRNAs are separated by internal transcribed spacers. We conclude that sequences corresponding to these spacers are removed post-transcriptionally from a high molecular weight pre-rRNA, resulting in a multiply fragmented large subunit rRNA. Internal transcribed spacers, in positions analogous to some of these additional Euglena rDNA spacers, have been found in the rDNA of other organisms and organelles. This finding supports the view that at least some internal transcribed spacers may have been present at an early stage in the evolution of rRNA genes.