In order to examine the possible involvement of mu-calpain in methylmercury (MeHg)-induced neurotoxicity in developing cortical neurons, we performed biochemical and immunohistochemical studies utilizing two antibodies which specifically recognize the 150-kDa mu-calpain-specific alpha-spectrin breakdown product (SBDP) and the active form of mu-calpain in rats on postnatal day 16. Soluble fractions of the cerebral cortex from control rats exhibited slight immunoreactivity for SBDP. Although the amount of SBDP in the cerebral cortex was only slightly increased the day after the final treatment of MeHg (10 mg/kg) for 3 or 7 consecutive days, there was a prominent accumulation of SBDP 3 days after the final treatment of MeHg for 7 consecutive days. On the other hand, the 76-kDa isoform of mu-calpain gradually increased after chronic treatment of MeHg, but markedly decreased 3 days after the final treatment of MeHg for 7 consecutive days. At this stage, many cortical neurons were densely stained with anti-SBDP antibody. The delayed increase in SBDP corresponded well with the delayed nature of the MeHg-induced neurotoxicity. When MK-801 (0.1 mg/kg), a non-competitive antagonist of N-methyl-D-aspartate (NMDA), was administered intraperitoneally with MeHg for 7 consecutive days, both neuronal damage and accumulation of SBDP were markedly depressed in the cerebral cortex 3 days after the final treatment. Our results indicate that mu-calpain activation and mu-calpain-mediated proteolysis of alpha-spectrin preceded neuronal damage in the developing cerebral cortex induced by chronic treatment of MeHg.