Neurons in the hippocampal formation vary markedly in their susceptibility to colchicine toxicity. The present study was directed at evaluating the effects of colchicine on the proteolytic breakdown of the cytoskeletal protein spectrin within the hippocampus in the rat. Quantified by immunoblot analysis of spectrin breakdown products, the extent of proteolysis was found to correlate with the relative vulnerability of different hippocampal subfields to colchicine toxicity. Levels of breakdown products increased dramatically between 1 and 2 days after colchicine injection, peaked between 2 and 4 days, and remained detectably elevated for at least 35 days. Two days after colchicine injection, the spectrin breakdown products were significantly more concentrated in the molecular layer than in the granule cell/hilar region of the dentate gyrus. The colchicine-induced increase in spectrin breakdown products was significantly reduced by pretreatment with the protease inhibitor leupeptin and was significantly elevated by pretreatment with the lysosomal inhibitor chloroquine. Immunohistochemical analyses of the hippocampus at various times after colchicine injection revealed changes in the distribution of spectrin-like immunoreactivity that paralleled the changes observed by Western blot analysis. Thus increased staining was observed in the molecular layer of the dentate gyrus at 2 and 4 days after the injection, while staining in CA3 was only slightly increased. In addition, abnormal staining of reactive astrocytes was prominent at 2 days. The mechanism whereby colchicine results in neuronal death is as yet unknown. However, the results presented here demonstrate that extensive proteolysis of a cytoskeletal protein occurs in response to the drug, suggesting a plausible mechanism for its neurotoxicity. The protease responsible for the effect is likely to be calpain since the process is non-lysosomal, leupeptin-sensitive and produces spectrin breakdown products indistinguishable from those generated by calpain treatment in vitro. These data support the hypothesis that calpain-mediated degradation of cytoskeletal elements is a common and early response to neurodegenerative events and serves as a trigger in the development of various neuropathologies.