In organello labelling of Trypanosoma brucei mitochondrial (mt) RNA was characterised with respect to nucleotide requirements and drug sensitivity. Mitochondrial transcriptional activity is maximal in the presence of all ribonucleoside-triphosphate NTPs, and can be inhibited by UTP depletion. Mitochondrial transcription can also be partially inhibited by actinomycin D (actD) or ethidium bromide (EtBr). Post-transcriptional UTP incorporation is insensitive to actinomycin D or ethidium bromide. Proteins were identified that interact with transcriptional and post-transcriptionally labelled RNAs, and confirm the in vitro RNA-binding properties discovered for a number of T. brucei mt proteins. These experiments reveal new strategies for studying mt transcription and processing in T. brucei mitochondria.