The spatial and temporal response of the cerebral microcirculation to mild hypercapnia was investigated via two-photon laser-scanning microscopy. Cortical vessels, traversing the top 200 microm of somatosensory cortex, were visualized in alpha-chloralose-anesthetized Sprague-Dawley rats equipped with a cranial window. Intraluminal vessel diameters, transit times of fluorescent dextrans and red blood cells (RBC) velocities in individual capillaries were measured under normocapnic (PaCO2= 32.6 +/- 2.6 mm Hg) and slightly hypercapnic (PaCO2= 45 +/- 7 mm Hg) conditions. This gentle increase in PaCO2 was sufficient to produce robust and significant increases in both arterial and venous vessel diameters, concomitant to decreases in transit times of a bolus of dye from artery to venule (14%, P < 0.05) and from artery to vein (27%, P < 0.05). On the whole, capillaries exhibited a significant increase in diameter (16 +/- 33%, P < 0.001, n = 393) and a substantial increase in RBC velocities (75 +/- 114%, P < 0.001, n = 46) with hypercapnia. However, the response of the cerebral microvasculature to modest increases in PaCO2 was spatially heterogeneous. The maximal relative dilatation (range: 5-77%; mean +/- SD: 25 +/- 34%, P < 0.001, n = 271) occurred in the smallest capillaries (1.6 microm-4.0 microm resting diameter), while medium and larger capillaries (4.4 microm-6.8 microm resting diameter) showed no significant changes in diameter (P > 0.08, n = 122). In contrast, on average, RBC velocities increased less in the smaller capillaries (39 +/- 5%, P < 0.002, n = 22) than in the medium and larger capillaries (107 +/- 142%, P < 0.003, n = 24). Thus, the changes in capillary RBC velocities were spatially distinct from the observed volumetric changes and occurred to homogenize cerebral blood flow along capillaries of all diameters.