To delineate the potential mechanism of neuroprotective effects of potassium channel openers we have investigated, how Ca(2+)-activated large conductance potassium channel (BK(Ca) channel) openers influence the production of reactive oxygen species (ROS) by rat brain mitochondria, since mitochondrial generation of ROS is known to have a crucial influence on neuronal survival. We studied the effects of BK(Ca) channel openers CGS 7184 and NS 1619 on hydrogen peroxide production rate of isolated rat brain mitochondria. In K(+)-containing media 3 microM of both channel openers reduced the hydrogen peroxide production rates by approximately 20%. This effect was not observed in Na(+)-containing media. This potassium-dependent partial inhibition of hydrogen peroxide production was found to be sensitive to the selective blockers of BK(Ca) channel iberiotoxin and charybdotoxin applied in nanomolar concentrations. Taken together, our data are compatible with the viewpoint that the opening of a Ca(2+)-activated large conductance potassium channel being localised in the inner membrane of brain mitochondria inhibits ROS production by respiratory chain complex I. This finding is suggested to explain the beneficial effects of BK potassium channel openers on neuronal survival.