The lbi (lipopolysaccharide biosynthesis interfering) RNA of phage Acm1, an untranslated RNA transcript of 97 nucleotides, previously shown to affect O-polysaccharide biosynthesis in various Escherichia coli strains, was found to downregulate the synthesis of the D-galactan II component of the O-specific polysaccharide in Klebsiella pneumoniae serotype O1. Enzymatic and Pb2+ probing experiments revealed that lbi RNA consists of two consecutive stem-loop structures, the 5'-proximal hairpin loop of 15 nucleotides being particularly accessible to single strand-specific probes. Based on the assumption that the 5'-proximal hairpin loop may be involved in an antisense interaction with cellular target RNAs, we randomly mutagenized one or two of its central nucleotides. Expression of mutated lbi RNA variants in K. pneumoniae serotype O1 relieved at least partly the repression of D-galactan II formation. In addition, a truncated version of lbi RNA lacking the 3'-proximal hairpin loop was almost as efficient as the wild-type RNA in downregulating D-galactan II synthesis. The results obtained indicate that the 5'-proximal hairpin loop of lbi RNA functions as a key structural element in the mechanism leading to the inhibition of D-galactan II biosynthesis in K. pneumoniae serotype O1.