We mapped the 5' UTR for five long hypothetical orfs from Trypanosoma cruzi; each one having a length of more than 10,000 bp. Our aim was to verify the constraints to the length of the 5' UTR and to identify the sites of alternative trans-splicing in the epimastigote stage of three T. cruzi strains. We used reverse transcription PCR to amplify the 5' UTR and demonstrated the transcription of all selected genes as well as additional trans-splicing sites in two of these genes. We observed that the length of the 5' UTR in these genes has a limit, in contrast to previous reports that indicated a trend for longer genes to display a proportionally long 5' UTR. The maximum length of the 5' UTR for the long genes analyzed in the present work is approximately 3% of the orf and, on average, is 1% of the orf length. The poly-pyrimidine tracts used as trans-splicing signal are in the range of 17-53 bases within a distance of 6-59 nt to first spliced-leader acceptor site. T. cruzi populations may use both signals differentially. We conclude that the limit for the 5' UTR length in long genes is determined primarily by the distance to neighboring genes.