Microglial cells, the macrophages of the brain, play an essential role in the propagation of neuroinflammation. Increased microglial cell migration in response to specific chemoattractants has been documented, but less is known about the differences between these stimuli and the signal transduction pathways that mediate their effects. Current methods to measure cell migration are often labor‐intensive and rely on the manual counting of cell number, so more efficient and objective methods are needed. Here we present an improved and higher‐throughput Boyden chamber technique that measures microglial cell migration by using DRAQ5, a nuclear dye that emits in the near‐infrared. Out of a panel of chemoattractants tested, we found that ATP and C5a potently stimulate the migration of mouse primary microglial cells. The stimulatory effects of ATP and C5a displayed significant additivity, suggesting that each chemoattractant stimulated migration through independent molecular mechanisms. Accordingly, we found key differences in these responses: ATP stimulated a combination of both chemokinesis and chemotaxis, and this response was mediated by the ROCK signaling pathway; whereas C5a stimulated only chemotaxis and this response was mediated by the Rac1 signaling pathway. Finally, we found that functional PI3‐kinase is only required for random basal microglial cell migration. Thus, our results show that distinct nonoverlapping signal transduction pathways control different modes of microglial cell migration and suggest that the targeting of these distinct molecular mechanisms should modulate different aspects of neuroinflammation propagation. © 2008 Wiley‐Liss, Inc.