Midazolam has been demonstrated to preserve the response of cerebral blood flow to CO2. However, the responsiveness of cerebral vessels or microcirculation during midazolam administration related to alteration of cerebral blood flow has not been explored. The purpose of this study was to examine the effects of midazolam on cerebral microcirculation. Nine cats were paralyzed and mechanically ventilated under nitrous oxide/oxygen anaesthesia. Using the closed cranial window technique and laser Doppler flowmetry, diameter of pial vessels and regional cerebral blood flow (rCBF) were examined on the surface of the cerebral cortex which was perfused by the middle cerebral artery. Before midazolam administration, haemodynamic variables, blood gases, rCBF, and diameter of pial vessels were determined as a control under normocapnia, hypocapnia, and hypercapnia conditions. After midazolam administration, given initially at 0.8 mg.kg-1.min-1 for 10 min and subsequently at 0.04 mg.kg-1.min-1 (total dose 10 mg.kg-1), the same variables were again analyzed. With regard to CO2 responsiveness, an 8.85% increase in rCBF was demonstrated for a Paco2 elevation of 1 kPa before midazolam administration, compared with a 7.47% increase after midazolam administration. With regard to the correlation between CO2 response and vessel diameter, arterioles less than 50 microns in diameter were more sensitive than those more than 50 microns in diameter, although there were no significant differences before or after midazolam administration. We conclude that CO2 responsiveness is preserved in terms of rCBF and vessel diameter after high doses of midazolam (10 mg.kg-1) in cats.