We took advantage of a subtractive hybridization procedure to isolate a set of cDNA clones of nodule-specific genes (nodulin genes) from developing soybean root nodules. Single-stranded 32P-labelled cDNA synthesized from nodule poly(A)+ RNA was hybridized with a large excess of uninfected root poly(A)+ RNA. Unhybridized cDNA was selected and used to screen nodule cDNA libraries. By this procedure we isolated several novel nodulin cDNA clones together with most of the nodulin cDNAs previously described. Four novel nodulin genes, which were expressed long before the onset of nitrogen fixation, were further characterized. GmN#36 and GmN#93 transcripts appeared in the roots less than 3 days after sowing and inoculation with Bradyrhizobium, but GmN#36 transcripts were also detected at very low levels in the stems of uninfected plants. Transcripts of GmN#315 and GmN#70 first appeared at 6-7 days, just before nodule emergence. Amino acid sequences of the predicted products of GmN#36, GmN#93 and GmN#70 exhibited no significant homology to proteins identified so far. The GmN#315 encoded protein has a limited but significant homology to some plant cyanins, suggesting that it is a metal-binding glycoprotein. In situ hybridization studies revealed that GmN#36 transcripts first appeared in the pericycle cells of the root stele near the infected site. During nodule emergence they were found in a few cell layers surrounding the vascular strands connecting the nodule meristem with the root stele, and in mature nodules they were present specifically in the pericycle cells in vascular bundles. These observations led us to hypothesize that GmN#36 gene products play a role in the transport and/or degradation of photosynthate. On the other hand, GmN#93 transcripts first appeared in the primary nodule meristem just below the root epidermis. In mature nodules they were only present in the infected cells.