Microglia are primary immune sentinels of the CNS. Following injury, these cells migrate or extend processes toward sites of tissue damage. CNS injury is accompanied by release of nucleotides, serving as signals for microglial activation or chemotaxis. Microglia express several purinoceptors, including a G_i-coupled subtype that has been implicated in ATP- and ADP-mediated migration in vitro. Here we show that microglia from mice lacking G_i-coupled P2Y₁₂ receptors exhibit normal baseline motility but are unable to polarize, migrate or extend processes toward nucleotides in vitro or in vivo. Microglia in P2ry₁₂^−/− mice show significantly diminished directional branch extension toward sites of cortical damage in the living mouse. Moreover, P2Y₁₂ expression is robust in the 'resting' state, but dramatically reduced after microglial activation. These results imply that P2Y₁₂ is a primary site at which nucleotides act to induce microglial chemotaxis at early stages of the response to local CNS injury.