Ribonuclease P (RNase P) is an essential enzyme that cleaves the 5' leader sequences of precursor tRNAs (pre-tRNAs) to generate mature tRNAs. The RNase P-like activity from Trypanosoma brucei mitochondria (mtRNase P) was purified over 10000-fold by sequential column chromatography. This is the first demonstration of such activity from mitochondria of parasitic protozoa. Its apparent molecular weight is approximately 70 kDa, considerably less than bacterial RNase P. Preliminary characterizations revealed no RNA component that is essential for this activity. Like other RNase P activities, the cleavage generates mature tRNAs with a terminal 5'-phosphate at the cleavage site and the 5' leader sequence with a 3'-hydroxyl. Disruption of the pre-tRNA tertiary structure inhibits the cleavage of the substrates. These data suggest that although all mitochondrial tRNAs are encoded in nuclear DNA in T. brucei, these cells contain an RNase P in the mitochondrion that cleaves the 5' terminal leader sequences of pre-tRNAs to generate mature tRNAs. Cleavage by mtRNase P of a pre-tRNA substrate that was divided into two fragments was demonstrated. This shows the feasibility of artificial regulation of gene expression that can be achieved by creating a complex made of target mRNA and a complementary small oligonucleotide that resembles natural substrates for RNase P.