Spliced leader (SL) RNA trans-splicing contributes the 5' termini to mRNAs in a variety of eukaryotes. In contrast with other trans-splicing metazoan groups (e.g., nematodes), flatworm spliced leader sequences are variable in both sequence and length in different flatworm taxa. However, an absolutely conserved and unique feature of all flatworm spliced leaders is the presence of a 3' terminal AUG. We previously suggested that the schistosome spliced leader AUG might serve as an initiator methionine for recipient mRNAs. Here we identified and examined trans-spliced cDNAs from a large set of newly available schistosome Ests and cDNAs. Twenty eight percent of the trans-spliced cDNAs have the SL AUG in frame with the major open reading frame of the mRNA. We identified over 40 cDNAs (40% of the SL AUG in frame clones) that require the SL AUG as an initiator methionine to synthesize phylogenetically conserved N-terminal residues characteristic of orthologous proteins. Furthermore, RNA transfection into different schistosome stages indicates that the flatworm SL AUG can serve as an initiator methionine in vivo. These data provide evidence that another function of spliced leader trans-splicing in flatworms is to provide some recipient mRNAs with an initiator methionine for translation initiation. We also examined and discuss the schistosome initiator methionine context and the effect of the spliced leader AUG added upstream and out of frame with the main open reading frame of recipient mRNAs on schistosome in vivo translation.