Polyadenylation is an important factor controlling RNA degradation and RNA quality control mechanisms. In this report we demonstrate for the first time that RNase R has in vivo affinity for polyadenylated RNA and can be a key enzyme involved in poly(A) metabolism. RNase II and PNPase, two major RNA exonucleases present in Escherichia coli, could not account for all the poly(A)-dependent degradation of the rpsO mRNA. RNase II can remove the poly(A) tails but fails to degrade the mRNA as it cannot overcome the RNA termination hairpin, while PNPase plays only a modest role in this degradation. We now demonstrate that in the absence of RNase E, RNase R is the relevant factor in the poly(A)-dependent degradation of the rpsO mRNA. Moreover, we have found that the RNase R inactivation counteracts the extended degradation of this transcript observed in RNase II-deficient cells. Elongated rpsO transcripts harboring increasing poly(A) tails are specifically recognized by RNase R and strongly accumulate in the absence of this exonuclease. The 39 oligo(A) extension may stimulate the binding of RNase R, allowing the complete degradation of the mRNA, as RNase R is not susceptible to RNA secondary structures. Moreover, this regulation is shown to occur despite the presence of PNPase. Similar results were observed with the rpsT mRNA. This report shows that polyadenylation favors in vivo the RNase R-mediated pathways of RNA degradation.