The localization of nitrogenase was studied in root nodules of Alnus incana (L.) Moench in symbiosis with a local source of Frankia. Nodules were fixed in glutaraldehyde and embedded in Epon or LR White. Ultrathin sections were examined by TEM after incubation with antisera against nitrogenase and gold-conjugated secondary antibodies. Antisera against dinitrogenase reductase (Fe-protein) from Rhodospirillum rubrum and dinitrogenase (MoFe-protein) from Azotobacter vinelandii were used. Immunogold label density in hyphae and in spores of Frankia was low and similar to that of plant tissue. Very young vesicles without septa showed a label density of Fe-protein similar to that of hyphae, while young vesicles with a few, usually non-transversing, septa had a significant but still low density of label. Mature vesicles, highly compartmentalised due to transversing septa, contained statistically significantly more Fe-protein label than all other cell types. Old degenerating vesicles always contained nitrogenase, about half as much as that of mature vesicles but often more than young vesicles. Similarly, MoFe label was not detected in hyphae, appeared in young but was greatest in mature vesicles. It is apparent that differentiation of vesicles reaches a certain stage of development prior to the onset of nitrogenase synthesis.