Direct in vivo evidence is still lacking for α4-integrin–mediated T cell interaction with VCAM-1 on blood-brain barrier–endothelium in experimental autoimmune encephalomyelitis (EAE). To investigate a possible α4-integrin–mediated interaction of encephalitogenic T cell blasts with VCAM-1 on the blood-brain barrier white matter endothelium in vivo, we have developed a novel spinal cord window preparation that enabled us to directly visualize CNS white matter microcirculation by intravital fluorescence videomicroscopy. Our study provides the first in vivo evidence that encephalitogenic T cell blasts interact with the spinal cord white matter microvasculature without rolling and that α4-integrin mediates the G protein–independent capture and subsequently the G protein–dependent adhesion strengthening of T cell blasts to microvascular VCAM-1.