Previous investigations have suggested that changes in platelet activity may play a key role in the pathophysiology of migraine via mechanisms involving the nitric oxide (NO) pathway. Changes in platelet response and nitrite levels have recently been demonstrated during migraine attacks, while there is considerable uncertainty about NO activity in headache-free periods. A reactive oxidant produced from NO and superoxide anion at the site of inflammation, peroxynitrite (ONOO-) has effects including changes in membrane activity and fluidity. The aim of the present study was to determine ONOO- levels in the platelets of patients suffering from migraine during the headache-free period. Nitric oxide synthase (eNOS and iNOS) expression in platelets and the effects of ONOO- on membrane Na+/K+-ATPase activity and membrane fluidity were also evaluated. Subjects were 57 patients suffering from migraine without aura and 35 controls. Blood samples were collected in the headache-free period. Platelet ONOO- levels were determined using dichlorofluorescein acetate with steady-state fluorescence. Platelets were then probed for induction of eNOS and iNOS expression by western immunoblotting. Membrane Na+/K+-ATPase activity and fluidity were determined with the fluorescent probes TMA-DPH and DPH. In the presence of extracellular l-arginine(100 micromol/l), ONOO- production was significantly greater in patients' platelets than in those of controls (P < 0.001). Western immunoblotting of platelet proteins evidenced higher iNOS expression in patients than in controls. In addition, platelet membrane Na+/K+-ATPase activity and membrane fluidity evaluated by TMA-DPH were significantly lower in patients (P < 0.001). In conclusion, migraine patients show intercritic changes in platelet membrane fluidity and activity that may be related to the oxidative stress caused by increased ONOO- levels.