In all organisms, tRNAs are matured by a series of post-transcriptional processing events before they can partake in protein synthesis. These include end trimming to generate the correct 5' and 3' ends, and a substantial number of post-transcriptional chemical modifications; these ensure proper structure and function. Despite much effort many modifications have not been recapitulated in vitro, which have made understanding their mechanism difficult, if not impossible. One such event entails the C to U editing of tRNAs at the anticodon, a discovery that dates back to almost 25 years, but one for which no in vitro assay was available. Recently, we established the first in vitro assay for C to U editing in any system. Our studies also revealed that before editing can take place, the edited position must also be methylated and both enzymes, the deaminase and the methylase, strictly require each other for activity. This allowed us to introduce a new concept of “enzyme co-activation”, one that may guide biochemical studies of other modifications for which no in vitro assay currently exist. Additionally, given that tRNA editing and modification in T. brucei is peppered with unique features, successful completion of these studies will generate important basic information on the mechanism and evolution of tRNA editing and modification, reveal unique features specific to T. brucei but which undoubtedly impact other systems. !