A mitochondrial specific RNase P is required to process 5′ leaders from mitochondrial tRNA precursors in Saccharomyces cerevisiae . Experiments with a pair of mitochondrial pretRNAs Asp having leaders of different base composition suggest that this enzyme is unexpectedly sensitive to leader sequence or structure. Asp-AU (75% AU leader) is cleaved by the mitochondria1 RNase P while Asp-GC (39% AU) is not. Both are substrates for E. coli RNase P. Partial nuclease digestions show that the tRNA portions of the two precursors differ in tertiary structure, while their 5′ leaders differ in secondary structure. It is unusual for an RNaseP to have substrate specificity requirements which preclude processing of a pretRNA known to be a suitable substrate for an RNaseP from another species.