Soybean nodulin-23 gene, induced in nodules formed due to symbiosis with Rhizobium, was found to contain multiple sequences capable of acting as eucaryotic and procaryotic promoters. The transcription start sites of this gene were localized by S1 nuclease mapping, primer extension with nodule mRNA and in vitro run-off transcription analysis. A major transcription start site was observed by S1 mapping; however, the the primer extension revealed a second start site. Sequence analysis showed the presence of tandemly arranged eucaryotic promoter sequences, each corresponding to one of the observed transcription start sites. In vitro transcription analysis with HeLa cell extract demonstrated the presence of another alpha-amantin-sensitive transcript ('anti-sense') with its direction opposite to that of the nodulin mRNA. The 5' ends for 80 nucleotides of nodulin and 'anti-sense' messages are complementary to each other. In addition, three tandemly arranged, functional procaryotic promoters (assayed in Escherichia coli) are found to be located within the eucaryotic promoter sites is similar to that of the major in vivo transcript in nodules. The potential significance for the presence of several promoter-like sequences in this gene is discussed.