Increasing evidence suggests a role for oxidative stress in several neurodegenerative diseases, including Alzheimer's disease (AD), and that selenium compounds may function as antioxidants. To evaluate the antioxidant mechanism of selenium, primary rat hippocampal neurons were pretreated with seleno-L-methionine (SeMet) for 16 h prior to treatment with iron/hydrogen peroxide (Fe(2+)/H(2)O(2)) or amyloid beta peptide (Abeta(2535)); free radical generation was assessed using laser confocal microscopy and CM-H(2)DCFDA and APF. Treatment with Fe(2+)/H(2)O(2) or Abeta significantly decreased cell survival and increased free radical generation compared to cultures treated with vehicle alone. In contrast, cultures pretreated with SeMet showed significantly (p < 0.05) increased survival and significantly lower CM-H(2)DCFDA and APF fluorescence compared to Fe(2+)/H(2)O(2) or Abeta treated cultures. To determine if SeMet protection was mediated by glutathione peroxidase (GPx), levels of GPx protein and activity were measured using confocal microscopy and a selenium-dependent GPx specific antibody and an activity assay. Pretreatment with SeMet significantly (p < 0.05) increased GPx protein and activity in Fe(2+)/H(2)O(2)- and Abeta-treated cultures compared to cultures treated with Fe(2+)/H(2)O(2) or Abeta alone. These data suggest that SeMet can decrease free radical generation induced by Fe(2+)/H(2)O(2) or Abeta through modulation of GPx and may be suitable as a potential therapeutic agent in neurodegenerative diseases where there is increased oxidative stress.