Three rat hybridoma cell lines have been isolated which produce monoclonal antibodies identifying a noduleenhanced, soluble component of Pisum sativum root nodules. These antibodies each recognized a protease-sensitive band (M(r) 95K) on SDS-polyacrylamide gels. The 95K antigen was resolved by isoelectric focusing into acidic and neutral components which were separately detected by AFRC MAC 236 and MAC 265 respectively. The third antibody (MAC 204) reacted with both acidic and neutral components through an epitope that was sensitive to periodate oxidation. These monoclonal antibodies were used for immunogold localizations at light and electron microscopic levels. In each case, the antigen was shown to be present in the matrix that surrounds the invading rhizobia in infection threads and infection droplets, as well as in the intercellular spaces between plant cell walls of nodules and also of uninfected roots. By contrast, a fourth monoclonal antibody, AFRC JIM 5, labelled a pectic component in the walls of infection threads, and JIM 5 was also found to label the middle lamella of plant cell walls, especially at three-way junctions between cells. The composition and structure of the infection thread lumen is thus comparable to that of an intercellular space.