Endoribonuclease RNase E has an important role in the processing and degradation of bacteriophage T4 and Escherichia coli mRNAs. We have undertaken a mutational analysis of the -71 RNase E processing site of T4 gene 32. A series of mutations were introduced into a synthetic T4 sequence cloned on a plasmid, and their effects on processing were analyzed in vivo. The same mutations were transferred into T4 by homologous recombination. In both the plasmid and the phage contexts the processing of the transcripts was similarly affected by the mutations. Partially purified RNase E has also been used to ascertain the effect of these mutations on RNase E processing in vitro. The hierarchy of the efficiency of processing of the various mutant transcripts was the same in vivo and in vitro. These results and an analysis of all of the known putative RNase E sites suggest a consensus sequence RAUUW (R = A or G; W = A or U) at the cleavage site. Modifications of the stem-loop structure downstream of the -71 site indicate that a secondary structure is required for RNase E processing. Processing by RNase E was apparently inhibited by sequences that sequester the site in secondary structure.