In the present study, we investigated whether or not the Abeta peptide itself spontaneously generates free radicals using electron spin resonance (ESR) spectroscopy while also observing the Abeta fibril formation by negative stain electron microscopy. The present results demonstrated a four-line spectrum in the presence of Abeta(1-40) with N-tert-butyl-alpha-phenylnitrone (PBN) but not in the presence of PBN alone in phosphate-buffered saline. Negative stain electron microscopy has shown that Abeta peptides after 96 h of incubation showed more amyloid-like fibrils than those after 72 h of incubation while the four-line spectrum obtained by ESR spectroscopy attained a maximum intensity after 72 h of incubation and thereafter its intensity immediately decreased during the 4-day incubation period. These results were also supported by a thioflavine-T (Th-T) fluorometric assay. In conclusion, the present results suggest that Abeta-associated free radical generation is correlated with Abeta fibril formation while its generation is only observed transiently during the process of Abeta fibril formation.