A putative promoter element responsible for transcription of the spliced leader (SL) gene of Trypanosoma cruzi was identified by overlapping deletion and linker scanning analyses of the upstream flanking sequences using the bacterial chloramphenicol acetyltransferase (CAT) gene as a reporter in transient transfections of cultured epimastigotes. Deletion or substitution of a proximal sequence element (PSE) between positions -53 and -40 relative to the transcription start point eliminated CAT gene expression. Comparison of SL genes from several strains of T. cruzi revealed two alternative sequence patterns for the putative SL PSE, both composed of a short run of purines followed by a run of pyrimidines. Moreover, an examination of these sequences supports the subdivision of T. cruzi isolates into two divergent groups. Double-stranded oligonucleotides containing the sequence of the PSE exhibited specific gel mobility shifts after incubation with T. cruzi nuclear extracts, suggesting that a transcription factor binds this site. Finally, experiments designed to increase the level of CAT expression from the SL promoter suggest that it is not a strong promoter in cultured T. cruzi epimastigotes.