In vitro transcription systems are a classic means to dissect mechanisms of gene expression at the molecular level. To begin an analysis of the biochemistry of gene expression in trypanosomes, we established an in vitro transcription system from cultured insect forms of Trypanosoma brucei. As a model we used the U2 snRNA gene which in vivo is transcribed by an RNA polymerase with characteristics of animal RNA polymerase III. To obtain maximum sensitivity in our assay, we adapted the so-called G-less cassette approach to the U2 snRNA gene promoter. Since an intragenic control region is required for accurate expression in vivo, we generated a series of mutations to substitute all guanosine residues in the intragenic control region. These mutants were shown to retain full transcriptional activity in vivo after transient expression in insect-form trypanosomes. In a cell-free extract, synthesis of the U2 G-less cassette RNA is correctly initiated, is mediated by RNA polymerase III as determined by RNA polymerase inhibitor studies, and is dependent on the integrity of the upstream B box element.