In conventionally-expressed eukaryotic genes, transcription start sites (TSSs) can be identified by mapping the mature mRNA 5'-terminal sequence onto the genome. However, this approach is not applicable to genes that undergo pre-mRNA 5'-leader trans-splicing (SL trans-splicing) because the original 5'-segment of the primary transcript is replaced by the spliced leader sequence during the trans-splicing reaction and is discarded. Thus TSS mapping for trans-spliced genes requires different approaches. We describe two such approaches and show that they generate precisely agreeing results for an SL trans-spliced gene encoding the muscle protein troponin I in the ascidian tunicate chordate Ciona intestinalis. One method is based on experimental deletion of trans-splice acceptor sites and the other is based on high-throughput mRNA 5'-RACE sequence analysis of natural RNA populations in order to detect minor transcripts containing the pre-mRNA's original 5'-end. Both methods identified a single major troponin I TSS located ∼460 nt upstream of the trans-splice acceptor site. Further experimental analysis identified a functionally important TATA element 31 nt upstream of the start site. The two methods employed have complementary strengths and are broadly applicable to mapping promoters/TSSs for trans-spliced genes in tunicates and in trans-splicing organisms from other phyla.