Transcription in vivo of small nuclear and cytoplasmic RNA genes of Trypanosoma brucei was previously shown to require the A and B blocks of a divergently transcribed tRNA or tRNA-like gene located approximately 100 nucleotides (nt) upstream. To understand the functioning of these transcription units, we have used the U6 snRNA/tRNA(Thr) genes as a model system. Saturation mutagenesis revealed that for transcription in vivo three elements are essential and sufficient. In addition to the previously described A and B boxes, sequences in the U6 coding region close to the 5' end participate in positioning RNA polymerase III at the start site, and thus constitute a third promoter element. We further showed that the function of the upstream A box, but not the B box, is strictly dependent upon its distance to the U6 gene internal control region. Using our recently developed transcription extract we further demonstrated that in vitro U6 transcription requires only the intragenic sequences and the upstream A box of the tRNA(Thr) gene. This apparent discrepancy between the in vivo and in vitro requirements is highly reminiscent of U6 snRNA gene transcription in the yeast Saccharomyces cerevisiae, and suggests the possibility that similar to the yeast system the B block of the trypanosome U6 snRNA gene promoter might be involved in chromatin organization.