Traditionally, new loci involved in the Rhizobium-legume symbiosis have been identified by transposon mutagenesis and/or complementation. Wide dispersal of the symbiotic loci in Rhizobium species NGR234, as well as the large number of potential host-plants to be screened, greatly reduces the efficiency of these techniques. As an alternate strategy designed to identify new NGR234 genes involved in the early stages of the symbiosis, we combined data from competitive RNA hybridisation, subtractive DNA hybridisation and shot-gun sequencing. On the assumption that the expression of most nodulation genes is triggered by compounds released by the host-plant, we identified, in the ordered cosmid library of the large symbiotic plasmid pNGR234a, restriction fragments that carry transcripts induced by flavonoids. To target genes not present in the closely related strain R. fredii USDA257, we selected fragments that also carried sequences purified by subtractive DNA hybridisation. Shot-gun sequencing of this subset of fragments lead to the identification of sequences with strong homology to diverse prokaryotic genes/proteins. Amongst these, a symbiotically active ORF from pNGR234a, is highly homologous to the leucine responsive regulatory protein of Escherichia coli (Lrp), is induced by flavonoids, and is not present in USDA257.