Our results indicate that RNase P has a very general role in the processing of tRNA precursors in E. coli, being responsible for the cleavage of virtually all precursor molecules at a site corresponding to the 5' end of the mature tRNA, and that at least two other RNases play specific roles in precursor processing. One of these, which may be RNase II, is responsible for removing extra nucleotides from the 3' end of tRNA precursors. The other, which we call RNase P2, is an endonuclease that cleaves precursors in spacer regions between different tRNA sequences; this enzyme is involved in the processing of large multimeric precursors.