mRNA maturation in Trypanosoma brucei depends upon trans-splicing, and variations in trans-splicing efficiency could be an important step in controlling the levels of individual mRNAs. RNA splicing requires specific sequence elements including conserved 5´ splice sites, branch points, pyrimidine-rich regions (poly(Y) tracts), 3´ splice sites (3´SS) and, sometimes, enhancer elements. To analyze sequence requirements for efficient trans-splicing, in the poly(Y) tract and around the 3´SS, we constructed a luciferase-b-galactosidase double-reporter system. By testing ~90 sequences, we demonstrated that the optimum poly(Y) tract length is ~25 nt. Interspersing a purely uridine-containing poly(Y) tract with cytidine resulted in increased trans-splicing efficiency, whereas purines led to a large decrease. The position of the poly(Y) tract relative to the 3´SS is important: an AC dinucleotide at position -3 and -4 can lead to a 20-fold decrease in trans-splicing. However, efficient trans-splicing can be restored by inserting a second AG dinucleotide downstream, which does not function as splice site but may aid in recruitment of the splicing machinery. These findings should assist the development of improved algorithms for computationally identifying 3´SS, and help to discriminate non-coding open reading frames from true genes, in current efforts to annotate the T. brucei genome.