It is unclear to what extent oxygen diffusion pathways through the cortex of the nitrogen-fixing zone of indeterminate nodules are liquid filled and whether a blockage of these pathways is involved in varying nodule oxygen permeability to control nitrogenase activity. We examined the proportion of water-filled intercellular spaces of lucerne (Medicago sativa L.) nodules with cryo-scanning electron microscopy. This technique allows for direct observation of water accumulation. Thirty percent of all intercellular spaces in the inner cortex of lucerne nodules were liquid filled. Decreasing the nodule oxygen permeability by detopping of the plant or by increasing the rhizospheric oxygen partial pressure to 80 kPa had no statistically significant effect on the water distribution in the intercellular spaces. Therefore, the hypothesis of a continuous aqueous diffusion barrier in the inner cortex could not be supported. The abundance of glycoproteins in intercellular spaces of the inner cortex was investigated with immunoelectron microscopy. No alteration due to detopping or after increase of the rhizospheric oxygen partial pressure was observed. Therefore, our results do not support the hypothesis of a short-term regulation of oxygen permeability by blockage of diffusion pathways through morphological changes in the cortex region of the nitrogen-fixing zone of lucerne nodules.