Five regions of the Bradyrhizobium japonicum genome, which are transcribed at high levels in nitrogen-fixing soybean (Glycine max) nodules, were identified. None of these regions contained previously identified genes (e.g., nif, nod, and fix genes) that are known to be essential for development of functional nitrogen-fixing nodules. To assess the role of these regions in the development of the B. japonicum-soybean symbiosis, we cloned and used them to construct B. japonicum strains, in which large DNA segments (2.0-6.8 kilobases) containing the highly transcribed regions were deleted. The deletion strains were examined for symbiotic effectiveness and were found to be indistinguishable from the wild-type strain. Transcription of the cloned regions under a variety of physiological conditions and in several defined mutant B. japonicum strains was also examined. The transcriptional start sites for one pair of divergent transcripts were determined; the promoters do not contain any of the conserved sequences found in B. japonicum genes involved in symbiosis or nitrogen metabolism.