In the mouse cortex, microglial cells respond vividly to acute injuries of the grey matter in an ATP-dependent manner. We now used 2-photon laser-scanning microscopy in mice with EGFP-labelled microglia (MG) to study the interaction of purinergic and nitric oxide (NO) signalling systems in the spinal white matter. In anaesthetized mice a laminectomy was performed at segment L4. Then MG responses to acute laser induced micro-injuries were recorded at high temporal and spatial resolution. Both, ATP and the volatile transmitter NO, tested as the NO-donor SPNO, attracted MG processes when locally applied in the spinal cord by a micropipette. However, both compounds could also act as suppressors of the MG response when the spinal cord below the laminectomy was uniformly incubated with the drugs. To analyse the interference between purinergic and NO-pathways, we combined the application of activating and blocking compounds for the two pathways. MG attraction induced by local intraspinal ATP-application was not reduced when the spinal cord was superfused with the guanylate cyclase blocker ODQ or SPNO. In contrast, the MG response to local SPNO application was reduced when ambient ATP was removed by the ATPase apyrase; but, the MG response to local SPNO-application was increased when the spinal cord was superfused with ATP. We conclude that ATP exerts its effect on MG independently of the NO-pathway, while components of the purinergic signal cascade modulate the MG attraction by NO.