We have investigated the functional role of lactate dehydrogenase-5 (LDH-5) as a helix destabilizing protein in regenerating rat liver. This study was based on the previous reports suggesting that helix destabilizing proteins isolated from rat liver and pheochromocytomal tumor cells were structurally and functionally similar to LDH-5. Additionally the accumulation of LDH A-mRNA was temporally well related to the onset time of - DNA replication during regeneration of rat liver. In this report, we aimed to further study in vivo system for the presence of LDH A-subunit in rat liver nuclear fraction and the temporal accumulation of LDH A-subunit in rat liver nuclei during regeneration period of rat liver. With the results we obtained, we present in this report several experimental findings supporting for the above notion. 1. The increased activity of LDH A-isozyme during regeneration of rat liver was not due to the enhanced glycolytic activity which was shown to be rather diminished in regenerating rat liver. 2. LDH A-subunit was identified as a tightly DNA bound form in hepatocyte nuclei after 18-hr in post-hepatectomy. 3. Electron microscope immunogold assay revealed that the temporal accumulation of LDH A-subunit in hepatocyte nuclei were well matched with the onset time of DNA synthesis in regenerating rat liver. These observations strongly support for the previous hypothesis suggesting that LDH A-isozyme may play a role as a bifunctional protein; that is, as a glycolytic enzyme and ssDNA-binding protein as well.