Adenosine-to-inosine editing in the anticodon of tRNAs is essential for viability. Enzymes mediating tRNA adenosine deamination in bacteria and yeast contain cytidine deaminase-conserved motifs, suggesting an evolutionary link between the two reactions. In trypanosomatids, tRNAs undergo both cytidine-to-uridine and ade- nosine-to-inosine editing, but the relationship between the two reactions is unclear. Here we show that down-regulation of the Trypanosoma brucei tRNA-editing enzyme by RNAi leads to a reduc- tion in both C-to-U and A-to-I editing of tRNA in vivo. Surprisingly, in vitro, this enzyme can mediate A-to-I editing of tRNA and C-to-U deamination of ssDNA but not both in either substrate. The ability to use both DNA and RNA provides a model for a multispecificity editing enzyme. Notably, the ability of a single enzyme to perform two different deamination reactions also suggests that this enzyme still maintains specificities that would have been found in the ancestor deaminase, providing a first line of evidence for the evolution of editing deaminases.