Opening of ATP-sensitive potassium (KATP) channels has been demonstrated to exert significant neuroprotection in in vivo and in vitro models of Parkinson's disease (PD), but the exact mechanism remains unclear. In the present study, various KATP channel openers (KCOs) sensitive to diverse KATP subunits were used to clarify the protective role of KATP channel opening in 1-methyl-4-phenylpyridinium (MPP+)-induced oxidative stress injury in mouse primary cultured mesencephalic neurons. The results showed that pretreatment with nonselective KCO pinacidil (Pin) or diazoxide (Dia), a KCO sensitive to Kir6.2/SUR1 KATP channels, protected mesencephalic neurons, especially dopaminergic neurons, against MPP+-induced injury in a concentration-dependent manner. However, cromakalim (Cro), an opener of Kir6.1/SUR2 but not Kir6.2/SUR1 KATP channels, failed to protect against MPP+-induced cytotoxicity. Furthermore, Pin and Dia but not Cro significantly suppressed the elevation of reactive oxygen species (ROS) triggered by MPP+ and prevented the loss of mitochondrial member potential (ΔΨm) and the release of mitochondrial cyotchrome c. Consequently, opening of KATP channels expressed in neurons could protect primary mesencephalic neurons against MPP+-induced cytotoxicity via inhibiting ROS overproduction and subsequently ameliorating mitochondrial function. © 2009 Wiley-Liss, Inc.