In members of the Trypanosomatidae family of parasitic protozoa, the mini-exon (MX) genes encode the mini-exon donor RNA (medRNA) that contributes a small, 39-nt exon to all pre-mRNAs during mRNA maturation. Previously we have shown that a single copy of a MX gene can be expressed continuously from a stable episome transfected into the monogenetic trypanosomatid Leptomonas seymouri. We now identify components of the MX gene promoter. A series of 10-bp block substitution mutations in a tagged MX gene were transfected into Leptomonas on an episomal vector. Expression of tagged and endogenous medRNA was assessed in stably transformed clonal cell populations. Results show that less than half of the 757-bp MX gene is necessary for medRNA transcription and that the key components of the MX gene promoter lie within the proximal 70-bp sequence upstream from the transcription initiation site. Transcription requires several sequence-specific blocks within this 70-bp region. Leptomonas cell extracts contain protein(s) that appear to interact with a subset of these sequences in gel mobility shift assays. All trypanosomatid MX genes contain an AT-rich region at the +10 to +20 position within the transcribed region of the MX gene. Mutagenesis of this region within an episomal copy of the MX gene did not block tagged medRNA synthesis but did cause a 10-fold increase in the steady-state amount of endogenous medRNA.