Macrophages are guided to sites of inflammation by chemoattractant gradients. These cells move by generating membrane protrusions in the direction of movement and retracting the trailing end. In a chemotactic gradient, macrophages need to direct the dominant membrane protrusion towards the source of chemoattractant. Chemoattractants such as complement component C5a are typically linked to PLC-beta and internal Ca2+ release, and possibly also to the Rho subfamily (RhoA, RhoB and RhoC) of Rho-GTPases via Rho-GEFS (guanine nucleotide exchange factors). The Rho subfamily has been implicated in regulating both membrane protrusive activity and tail retraction. The role of Ca2+ signaling is unclear,although so-called Ca2+ flickers at the leading edge have been implicated in gradient sensing. Here we investigated the role of Rho subtypes in chemotaxis and tail retraction using mouse knockout models, and we probed the role of internal Ca2+ release using strategies to deplete internal stores.