For high throughput screening of root nodule-enhanced genes, cDNA libraries specific for three different developmental stages of soybean root nodules were constructed after inoculation with Bradyrhizobium japonicum USDA110. 5,469 cDNA clones were sequenced and grouped into 2,511 non-redundant (nr) ESTs consisting of 769 contigs and 1,742 singletons. Using similarity searches against several public databases we constructed a functional classification of the ESTs into root nodule-specific nodulin genes, stress-responsive genes and genes related to carbon and nitrogen metabolism. We also constructed a cDNA microarray with 382 selected clones that appeared to be up-regulated in the root nodule. Using the microarray we compared the transcript levels of uninfected roots and root nodules from four developmental stages. We identified 81 genes that were differentially expressed, and grouped them into seven clusters according to the similarity of their expression profiles, using a hierarchical clustering algorithm. Clusters 1, 2, 3, and 6, comprised of 58 genes, showed root nodule-enhanced expression. The information from this study will be used to analyze the roles of root nodule-specific genes and signaling pathways during root nodule development.