Expression of eukaryotic nuclear encoded tRNA genes requires two transcription factors, TFIIIB and TFIIIC. To determine if the highly evolutionarily diverged parasitic protozoan Trypanosoma brucei possesses any analogous factors, trypanosome nuclear extracts were prepared. Using these extracts in gel-retardation assays on trypanosome tRNA genes we detected three specific protein-DNA complexes, a highly retarded species and a less retarded pair of complexes. Introduction of mutations into the B box of a lysyl tRNA gene greatly reduced formation of all three complexes. Footprinting studies indicated that all complexes protected the B box of the tRNA gene from cleavage. The most highly retarded complex protected both the A and B boxes from DNasel cleavage. The less retarded pair of complexes showed footprints on the B box alone and the lowest B box specific complex is shown to be resistant to the polyanion heparin. All three complexes are demonstrated to induce bends in the DNA on binding.