The temporal evolution of changes in the distribution of neurofilaments, vimentin-intermediate filaments (IF) and glial fibrillary acidic protein (GFAP)-IF induced by 2,5-hexanedione (2,5HD) has been investigated in dissociated cultures of fetal mouse spinal cord and dorsal root ganglia (DRG). Aggregates of GFAP-IF, labelled by antibodies against GFAP, appeared in astrocytes approximately one week after axonal swellings filled with neurofilaments were detected in neurons by labelling with antibodies to 68 kD or 200 kD neurofilament proteins. The threshold concentration required for aggregation of GFAP-IF was 1.4 to 2 times that required for neurofilament accumulations. In contrast to findings in cultured human skin fibroblasts, aggregates of vimentin-IF were not found in 2,5HD-treated cultures from mouse tissue (non-neuronal cells of spinal cord-DRG or fibroblasts from skin or muscle). The IF of 3T3 cells, a mouse fibroblast cell line, formed diffuse juxtanuclear aggregates only after high levels of exposure to 2,5HD. The sensitivity of vimentin-IF to aggregation by 2,5HD was proportional to the IF content of the cell type. These differences in the sensitivity of IF to aggregation by 2,5HD among cell types and species explain why IF-aggregates have not been observed previously in non-neuronal cells in human patients with the neuropathy associated with 2,5HD, or in experimental studies with rodents.