Differential expression of genes localized within the polycistronic puf operon of Rhodobacter capsulatus is partly due to altered stabilities of individual mRNA segments. We show that the 5' untranslated region (UTR) of pufB contributes to the unusual longevity of the 0.5 kb light-harvesting (LH) I specific pufBA mRNA and of the 2.7 kb pufBALMX mRNA. Three stem-loop structures have been identified within the pufQ-pufB intercistronic region by means of RNA secondary-structure analysis in vitro and in vivo. Deletion analysis of the pufB 5' UTR indicates that the complete set of secondary structures is required to maintain wild-type levels of pufBA mRNA stability. A phylogenetic comparison of pufB 5' UTRs of other photosynthetic bacteria reveals an evolutionary conservation of the base-pairing potential despite sequence divergence. Comparison of puf mRNA decay in Escherichia coli strains with or without endoribonuclease E (RNase E) activity suggests that the pufB 5' secondary structures protect the downstream mRNA segment against degradation by RNase E. Removal of the 117-nucleotide pufQ-pufB intercistronic region results in loss of stability for the pufBA and pufBALMX mRNAs with concomitant stabilization of the full-length puf primary transcript (QBALMX). We therefore conclude that the deleted sequence functions both as a stabilizing element for pufBALMX and pufBA segments and as a target site for initial rate-limiting decay of the unstable pufQBALMX mRNA.