The deafness-associated 7472insC mtDNA mutation was previously shown to decrease the steady-state level of tRNA(Ser(UCN)) post-transcriptionally. To identify the affected tRNA maturation step(s) we analysed the effects of the mutation on processing in vivo and in vitro. tRNA(Ser(UCN)) from cybrid cells homoplasmic for 7472insC contained a high frequency (>11%) of molecules misprocessed at one or both termini. In vitro assays using partially purified HeLa cell RNase P and mitochondrial tRNA 3' processing endonuclease (tRNase Z) confirmed that the efficiency of both 5' and 3' processing was impaired. A mutant precursor not already processed at the 5' end was poorly processed in vitro by tRNase Z. Misprocessing at the 3' end further impaired the efficiency and accuracy of 5' processing of the mutant substrate. The mutation thus appears to affect several distinct, but interdependent, RNA processing steps, with the predicted outcome dependent on the exact processing pathway operating in vivo.