Glial reactions may play a major role after spinal cord injury. Therefore, with two-photon laser-scanning microscopy we now studied the response of microglial cells to a laser lesion in the spinal dorsal columns of adult transgenic mice with microglial-specific EGFP expression. Under full anaesthesia (initially pentobarbital 80 mg/kg i.p., followed by methohexital-Na 50 mg/kg/h, i.v.) a laminectomy was performed over the spinal cord segment L4 and the mouse was placed under the microscope. Microglial cells displayed spontaneously highly motile branches that continuously surveyed the microenvironment and axonal structures. Within 1–2 min after laser lesions microglia started to send processes to the direction of the lesion. In the course of the following hours, microglial cells moved towards the lesion leading to a distinct local microgliosis. Superfusion of the spinal cord with non-specific and specific nitric oxide synthase and guanylate cyclase inhibitors (methylene blue and 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one) led to an almost complete blockade of the microglial response around the lesion site. Long lasting superfusion with the NO-donor spermine-NONOate (SPNO) also blocked the microglia response, while local intraspinal application of SPNO caused an attraction of microglia. The results indicate that early microglial reaction contributes essentially to the acute processes after spinal cord injury and that this early reaction involves the NO-system.