A full-length cDNA clone, designated Ljca1, coding for a beta-type carbonic anhydrase (CA; EC: 18.104.22.168) was isolated from a Lotus japonicus nodule cDNA library. Semi-quantitative RT-PCR analysis revealed that Ljca1 codes for a nodule-specific CA, transcripts of which accumulate at maximum levels in young nodules at 14 days post-infection (d.p.i.). In situ hybridization and immunolocalization revealed that Ljca1 transcripts and LjCA1 polypeptides were present at high levels in all cell types of young nodules. In contrast, in mature nodules both transcripts and polypeptides were confined in a few cell layers of the nodules inner cortex. However, the central infected tissue of both young and mature nodules exhibited high CA activity, indicating the presence of additional CA isoforms of plant and/or microbial origin. This was supported by the finding that a putative Mesorhizobium loti CA gene was transiently expressed during nodule development. In addition, the temporal and spatial accumulation of phosphoenolpyruvate carboxylase (PEPC; EC: 22.214.171.124) was determined by semi-quantitative RT-PCR and immunolocalization. The results suggest that LjCA1 might fulfill different physiological needs during L. japonicus nodule development.