In this work we performed in situ hybridization of a carbonic anhydrase RNA 35S-labeled probe on nodule sections previously embedded in a methacrylate resin. The results were more precise and reproducible than those obtained on paraffin-embedded nodules. Thanks to the small thickness of the sections and the good preservation of tissue during sectioning, the specific localization of the carbonic anhydrase gene expression in the inner cortex of the nodule could be described quite precisely. It is argued that these results are consistent with the hypothesis of osmoregulation of the symbiotic nitrogen fixation. Moreover, the quality of these results with a radioactive probe makes it possible to consider using ISH for such rhizospheric applications as localizing and quantifying a microbial invasion of the endorhizosphere or the rhizoplan by using bacterial- or fungal-specific RNA probes, and counting the number of radioactive dots per cell. However, major artifacts observed with a digoxegin RNA cold probe makes it necessary to recommend the use of a radioactive RNA probe for these prospects.