The location of the brain within the cranium has prevented in vivo studies with microscopical resolution for a long time. Cranial window techniques provide microscopical access to the brain surface, but subsurface structures cannot be visualized with conventional microscopy. Confocal microscopy with its increased depth of penetration and optical sectioning capabilities is ideally suited for the investigation of thick biological specimens. Due to the scanning process, however, temporal resolution is limited, a significant disadvantage in the in vivo setting. In this article we demonstrate that confocal laser scanning microscopy can be utilized to study brain cortex microvascular morphology, capillary hemodynamics, leukocyte behaviour and intracellular ion concentrations in anesthetized rats through a closed cranial window. Three different confocal microscopes are compared: a Biorad MRC-600 with multiline Kr/Ar-Laser (488/568/647 nm), a Noran Odyssey acousto-optic scanning microscope with multiline Ar-Laser (458/488/514/529 nm) and a Biorad Viewscan DVC-250 slit scanning microscope with Ar-Laser (488/514 nm). With all microscopes a Zeiss × 40 water immersion objective, n.a. 0.75 is used. A Laser-Doppler flowmeter continuously measures regional cerebral blood flow in the area of microscopical investigation. As fluorescent dyes we used: fluorescein sodium as blood plasma marker (given I.V.); rhodamine 6G to label leukocytes (given I.V.); and the AM-esters of BCECF (pH-sensitive), Fluo-3 and Calcium-Green (both calcium-sensitive) as intracellular ion-concentration markers (loaded via superfusion over the cranial window). With this setup, we are able to study the flow dynamics in the capillary network of the cortex (erythrocyte flow velocities and flux rates), the behaviour of leukocytes in capillaries and postcapillary veins (plugging of capillaries, adhesion to the endothelium, extravasation into the parenchyma), and intracellular changes of [H +] and [Ca 2+] under physiological and pathophysiological conditions (cerebral ischemia and meningitis). The comparison between the conventional CLSM (Biorad 600) and the real time CLSMs revealed that the increase in temporal resolution afforded by the real time instruments is offset by a reduction in spatial resolution and, most importantly, in the signal to noise ratio, resulting in a lower depth of penetration into the tissue and necessitating frame averaging.