Since the histidine-containing dipeptide carnosine (beta-alanyl-L-histidine) is believed to have many physiological functions in the brain, we investigated the neuroprotective effects of carnosine and its mechanisms of action in an in vitro model of neurotoxicity induced by N-methyl-d-aspartate (NMDA) in differentiated PC12 cells. Pretreatment with carnosine increased the viability and decreased the number of apoptotic and necrotic cells measured by MTT and Hoechst 33342 and propidium iodide (PI) double staining assays. Carnosine also can inhibit the glutamate release and increase HDC activity and the intracellular and extracellular contents of carnosine, histidine and histamine detected by high-performance liquid chromatography (HPLC). The protection by carnosine was reversed by alpha- fluoromethylhistidine, a selective and irreversible inhibitor of histidine decarboxylase (HDC). Pyrilamine and thioperamide, selective central histamine H(1) and H(3) antagonists also significantly reversed the protection of carnosine. Further, the inhibition of glutamate release by carnosine was reversed by thioperamide. Therefore, the protective mechanism of carnosine may not only involve the carnosine-histidine-histamine pathway, but also H(1)/H(3) receptors and the effective inhibition of glutamate release. This study indicates that carnosine may be an endogenous protective factor and calls for its further study as a new antiexcitotoxic agent.