We have localized the six ribosomal RNAs (rRNAs) which encode the 28S rRNA region of Trypanosoma brucei. These six rRNAs include two large rRNAs, 28S alpha (approx. 1840 nt) and 28S beta (approx. 1570 nt), and four small rRNAs of approximate sizes 220, 180, 140 and 70 nt. Three of these four small rRNAs (180, 70 and 140) are found at the 3' end of the 28S rRNAs region. Sequence analysis of this area shows that these three small rRNAs encode Domain VII, the last domain of secondary structure in the 28S rRNAs of eukaryotes. Hybridization of labeled nascent RNA to the cloned repeat unit and S1 nuclease protection analysis of putative precursors show that transcription initiates approximately 1.2 kb upstream of the 18S rRNA and terminates after the last small rRNA (140) at the 3' end of the 28S rRNA region. Analysis of three putative rRNA precursors suggests that the small rRNAs are not processed from the primary transcript until after the usual processing of the 5.8S rRNA region.