The DNA sequence which spans the internal transcribed spacers of a cloned ribosomal transcription unit from the sea urchin, Lytechinus variegatus, has been determined. The region extends from the conserved Eco RI site near the 3' end of the 18S rDNA to a Bam HI site in the 26S rDNA and includes 232 nucleotides coding for 18S rRNA, 367 nucleotides of internal transcribed spacer, 159 nucleotides coding for 5.8S rRNA, 338 nucleotides of internal transcribed spacer, and 505 nucleotides coding for 26S rRNA. The rRNA coding regions were identified by direct analysis of 3'-labeled 18S and 5.8S rRNA and 5'-labeled 5.8S rRNA, and by sequence homology of the 26S rDNA with yeast and vertebrate 26/28S rRNAs. The internal transcribed spacers are GC-rich, similar to those of vertebrates. The 5.8S and 5' 26S rDNA sequences support a proposed model for a structural domain of the yeast large subunit ribosomal RNA (Veldman et al. [1981] Nucleic Acids Res. 9, 6935-6952).