Ribonuclease E (RNase E) is known to play an important role in mRNA decay and RNA processing in Escherichia coli. While several substrates for RNase E have been identified, the specificity for the recognition and cleavage sites has not been completely determined. In this study, micF RNA, an antisense RNA found in E. coli and related bacteria, was found to be a substrate for RNase E in vitro. Two cleavage sites were mapped, and both are found in the sequence context UA/UUU and are located within 10 nucleotides upstream of stem-loop structures. These results help define a generalized RNase E cleavage/recognition pattern.