Earlier work has shown that RNase E cleaves RNAI, the antisense repressor of replication of ColE1-type plasmids, producing pRNAI-5, whose further decay is mediated by the poly(A)-dependent activity of polynucleotide phosphorylase and other 3' to 5' exonucleases. Using a poly(A) polymerase-deficient strain to impede exonucleolytic decay, we show that RNAI is additionally cleaved by RNase E at multiple sites, generating a series of decay intermediates that are differentially retained by the RNA binding domain (RBD) of RNase E. Primer extension analysis of RNAI decay intermediates and RNase T1 mapping of the cleavage products of RNAI generated in vitro by affinity-purified RNase E showed that RNase E can cleave internucleotide bonds in the bubble regions of duplex RNA segments and in single-stranded regions. Chemical in situ probing of a complex formed between RNAI and the RBD indicates that binding to the RBD destabilizes RNAI secondary structure. Our results suggest a model in which a series of sequential RNase E-mediated cleavages occurring at multiple sites of RNAI, some of which may be made more accessible to RNase E by the destabilizing effects of its RBD, generate RNA fragments that are further degraded by poly(A)-dependent 3' to 5' exonucleases.