A cell-free transcription system for the spliced leader (SL) RNA gene of the trypanosomatid Leptomonas seymouri has been developed. Accurately initiated transcription was achieved using cell extracts and a template in which the transcribed region of the SL RNA was replaced with a guanosine-less sequence (G-less cassette). The extract was also able to direct accurate initiation of RNA from an L. seymouri tagged U2 snRNA gene, which may be expressed via a transcriptional apparatus shared by the SL RNA gene. In vivo transcription analysis was used previously to define essential sequence components of the SL RNA gene promoter (Hartree D, Bellofatto V. Mol Biochem Parasitol 1995:71:27-39). A substitution mutation in the upstream promoter element (bp - 50 to - 70) markedly reduced transcription in vitro as did deletion of this and the middle promoter element (bp - 30 to - 40). Thus, the in vitro transcription system correctly responds to promoter mutations and is useful for investigating SL RNA and snRNA gene expression.