Characteristic accumulation of fibrous materials is well recognized in the brain with several neurodegenerative diseases. To elucidate the mechanism of the abnormal accumulation of fibrils in neurodegenerative diseases, the process of experimental neurofibrillary change (E-NFC) formation was studied. E-NFC, intraneuronal accumulation of 10 nm filaments, was induced by intracerebral injection of aluminium salt to New Zealand white rabbits. The changes in methabolism of neurofilament subunit protein was studied because E-NFC is known to be consisted of phosphorylated neurofilament protein. The synthesis, localization and phosphorylation of the protein during the process of E-NFC formation was studied by northern blotting, ELISA, confocal laser microscopy, and immunohistochemical procedures. The low molecular weight subunit of neurofilament protein (NF-L) and its mRNA was decreased in the cerebrum, cerebellum and brain stem. The heavy molecular weight subunit of neurofilament protein (NF-H) was increased by up to 5-fold and the ratio of NF-H to NF-L protein was increased up to about 30-fold in brain stem while its mRNA was increased by up to 2.5-fold. By confocal laser microscopy, localization of NF-H and NF-L was compared before and after intoxication, E-NFC was mainly composed of NF-H, disrupting the diffuse localization of NF-L and NF-H in the neuronal perikarya. By immunohistochemistry, non-phosphorylated NF-H was accumulated in perykarya as early as three day after intoxication, while phosphorylated NF-H was not. Five days after intoxication, the staining of non-phosphorylated NF-H around nucleus was decreased while the staining of phosphorylated NF-H appeared at proximal processes. Seven days after intoxication, the staining of non-phosphorylated NF-H was almost vanished while the staining of phosphorylated NF-H became stronger and more spread. Considering the levels of mRNA and protein of NF-L and NF-H, it is unlikely that the accumulation of non-phosphorylated NF-H in the early stage is due to up-regulation of neurofolament protein synthesis. Impaired axonal transport and/or suppressed protein degradation can be possibilities to account for the discrepancy between the protein and mRNA levels. The results indicate that the imbalance between NF-H and NF-L proteins leads to the up-regulation of phosphorylation of NF-H and the growing imbalance between the proteins, which will eventually result in the formation of E-NFC.