Polyadenylation plays an important role in regulating RNA stability in Trypanosoma brucei mitochondria. To date, little is known about the enzymes responsible for the addition of mRNA 3' tails in this system. In this study, we characterize a trypanosome homolog of the human mitochondrial poly(A) polymerase, which we term kPAP2. kPAP2 is mitochondrially localized and expressed in both bloodstream and procyclic form trypanosomes. Targeted gene depletion using RNAi showed that kPAP2 is not required for T. brucei growth in either bloodstream or procyclic life stages, nor is it essential for differentiation from bloodstream to procyclic form. We also demonstrate that steady state abundance of several mitochondrial RNAs was largely unaffected upon kPAP2 down-regulation. Interestingly, mRNA 3' tail analysis of several mRNAs from both life cycle stages in uninduced kPAP2 RNAi cells demonstrated that tail length and uridine content are both regulated in a transcript-specific manner. mRNA-specific tail lengths were maintained upon kPAP2 depletion. However, the percentage of uridine residues in 3' tails was increased, and conversely the percentage of adenosine residues was decreased, in a distinct subset of mRNAs when kPAP2 levels were down-regulated. Thus, kPAP2 apparently contributes to the incorporation of adenosine residues in 3' tails of some, but not all, mitochondrial mRNAs. Together, these data suggest that multiple nucleotidyltransferases act on mitochondrial mRNA 3' ends, and that these enzymes are somewhat redundant and subject to complex regulation.