Mature mRNAs of trypanosomatid protozoa result from the joining of at least two exons, which are initially transcribed as separate RNAs. In all trypanosomatids examined to date, the first exon (mini-exon) is encoded by approximately 200 tandemly reiterated genes. In characterizing the mini-exon genes of Leptomonas seymouri, we identified two predominant size classes of repetitive sequences that hybridized strongly to the L. seymouri mini-exon sequence. These two sequences are arranged as interspersed clusters. DNA sequence analysis of a clone representing the smaller size class demonstrated that these sequences have the capacity to encode a mini-exon donor (med)RNA corresponding to the 86 nt component seen in Northern blots of L. seymouri RNA. The larger size class comprises a family of related sequences, some of which contain DNA inserted into the mini-exon portion of the medRNA gene. The specific insert identified here (LINS 1) is exclusively associated with medRNA sequences, and is present in approximately 20% of the larger size class of L. seymouri medRNA genes. Disregarding the insertion, the sequences of the smaller bona fide mini-exon genes and the gene copy containing the insert were almost identical. The insert sequence is transcribed in the same direction as medRNA to yield at least four small non-polyadenylated RNAs, which appeared not to be linked to medRNA sequences.