Trypanosome mRNA is processed to maturity in a novel trans-splicing reaction during which a 35-nucleotide (nt) spliced leader (SL) is joined to the 5' ends of most structural gene transcripts. We have examined this process in Trypanosoma cruzi, the causative agent of Chagas' disease in Central and South America. In this communication, we characterize the genes encoding the SL (SL gene) in five different strains of T. cruzi by hybridization analysis and show that the genome of each of these strains contains numerous tandemly repeated copies of the SL gene. We demonstrate that the SL genes show remarkable intrastrain homogeneity, but significant interstrain heterogeneity. We have cloned and sequenced one of the SL repeats from T. cruzi strain CL and used synthetic oligodeoxyribonucleotides designed to hybridize to SL gene transcripts in Northern analyses of T. cruzi RNA to identify an approx. 110-nt putative SL primary transcript (SL-RNA). The 5' end of the SL-RNA was mapped to the first nt of the SL by primer extension analyses. The sequence of the 110-nt SL-RNA was used to generate a predicted secondary structure, and this structure compared favorably to the predicted secondary structures of SL transcripts of other trypanosomatids.