Ribonuclease E (RNAase E) was isolated in a complex that also contained polynucleotide phosphorylase (PNPase). Besides copurification, evidence for an association of these enzymes comes from sedimentation and immunoprecipitation experiments. Highly purified RNAase E correctly processed E. coli 5S ribosomal RNA, bacteriophage T4 gene 32 mRNA and E. coli ompA mRNA at sites known to depend on the rne gene for cleavage in vivo. The difference between previous smaller estimates of the size of RNAase E and that reported here apparently is due to the sensitivity of the enzyme to proteolysis during purification. The discovery of a specific association between RNAase E and PNPase raises the intriguing possibility that these enzymes act cooperatively in the processing and degradation of RNA.