The 5' sequences essential for spliced leader (SL) addition to RNA encoding the abundant Leishmania surface protease gp63 were determined. A DNA segment found upstream of all Leishmania chagasi gp63 genes was cloned in front of a luciferase gene, and luciferase activity was measured in transiently transfected L. chagasi promastigotes. Two hundred and twenty bp of the upstream region was needed for optimal luciferase activity. Deletions and point mutations were placed in this segment to determine which nucleotides participate in the splicing events. A region containing 87% pyrimidines located between 31 and 69 bp upstream of the splice acceptor dinucleotide and containing three potential branch point A residues was highly beneficial for reported gene activity. In contrast, a 30-nt pyrimidine-rich sequence immediately upstream of the splice acceptor site did not by itself enhance luciferase activity. Luciferase activity was associated with the presence of trans-spliced luciferase mRNA. The results suggest that sequences 31-69 bp upstream of gp63 genes enhance gene expression through provision of signals for efficient trans-splicing.