Symbiosome membranes (SM) and symbiosome space (SS) fractions were isolated from nitrogen fixing soybean root nodules and analyzed using non-gel proteomic techniques. Bicarbonate stripping and chloroform-methanol extraction of isolated SM were used to enrich for hydrophobic integral membrane proteins.. One-hundred and seventy-two proteins were identified as components of the SM, with an additional fifteen proteins identified from peripheral membrane and SS protein fractions. Proteins involved in a range of cellular processes such as metabolism, protein folding and targeting, signalling and transport were identified. These included a number of proteins previously localized to the SM, such as nodulin 26, an aquaglyceroporin. Transport classes identified include sulphate, calcium, peptide/dicarboxylate, nitrate and metal ion transporters. This proteome will provide a rich resource for the study of the legume-rhizobium symbiosis.