RNA editing in kinetoplastid protists is required for the maturation of mitochondrial pre-mRNAs and occurs by protein-catalyzed cycles of uridylate insertion and deletion. During the complex life cycle of Trypanosoma brucei this process is differentially regulated in the mammalian bloodstream and insect procyclic stages. Complementary guide RNAs (gRNAs) direct editing, but the abundance of these transcripts is not developmentally controlled. The establishment of in vitro systems that recreate efficient RNA editing in bloodstream T. brucei would be valuable for mechanistic studies of regulation. Here we describe a robust in vitro system that reconstitutes full cycles of both U insertion and U deletion in bloodstream trypanosomes, and the first direct comparisons of the in vitro systems for strains of mammalian and insect stages.