We used confocal laser scanning microscopy (CLSM) to investigate the morphology and three-dimensional relationships of the microcirculation of the superficial layers of the rat brain cortex in vivo. In anesthetized rats equipped with a closed cranial window (dura mater removed), after i.v. injection of 3 mg/100 g of body weight of fluorescein in 0.5 ml of saline, serial optical sections of the brain cortex intraparenchymal microcirculation were taken. Excitation was at a wavelength of 488 nm (argon laser), and emission was collected above 515 nm. CLSM provided images of brain vessels with sufficient signal-to-noise ratio for three-dimensional reconstructions down to a depth of 250 microns beneath the surface of the brain. Compared to conventional fluorescence microscopy, CLSM has a much higher axial resolution and higher depth of penetration. Laser light-induced intravascular aggregates, irregularities of erythrocyte flow, or microvascular occlusions ("light and dye injury") were not apparent in the current experimental paradigm. CLSM is a promising new tool for in vivo visualization of the cerebral microcirculation. Future studies have to characterize the potential damage to the tissue dye mechanisms.