Nuclear Dictyostelium discoideum RNase P was subjected to vigorous deproteinization procedures. After treatment with proteinase K followed by phenol extraction of samples containing D. discoideum RNase P activity, a new enzymatic activity was recovered. The proteinase K/phenol/SDS treated enzyme cleaves Schizossacharomyces pombe tRNAser (supS1), D. discoideum tRNASer and tRNALeu precursors several nucleotides upstream of the cleavage site of RNase P, liberating products with 5'-hydroxyl ends. This activity seems to be associated with one or two RNA molecules copurifying with D. discoideum RNase P activity as judged by its inhibition in the presence of micrococcal nuclease, which is in contrast to its resistance to proteinase K/phenol/SDS treatment.