Prokaryotic regulatory small RNAs act by a conserved mechanism and yet display a stunning structural variability. In the present study, we used mutational analysis to dissect the functional anatomy of RybB, a σE-dependent sRNA that regulates the synthesis of major porins in Escherichia coli and Salmonella. Mutations in the chromosomal rybB locus that altered the expression of an ompC–lac fusion were identified. Some of the mutations cluster within a seven-nucleotide segment at the 5′ end of the sRNA and affect its ability to pair with a sequence 40 nucleotides upstream from ompC translation start site. Other mutations map near the 3′ end of RybB, destabilizing the sRNA or altering its binding to Hfq. The 5′ end of RybB is also involved in ompD regulation. In this case, the sRNA can choose between two mutually exclusive pairing sites within the translated portion of the mRNA. Some of the RybB 5′ end mutations affect the choice between the two sites, resulting in regulatory responses that diverge from those observed in ompC. Further analysis of RybB target specificity identified chiP (ybfM), a gene encoding an inducible chitoporin, as an additional member of the RybB regulon. Altogether, our results indicate that an heptameric ‘seed’ sequence is sufficient to confer susceptibility to RybB regulation.