T cells need to cross endothelial barriers during immune surveillance and inflammation. This involves T-cell adhesion to the endothelium followed by polarization and crawling with a lamellipodium at the front and contractile uropod at the back. T cells subsequently extend lamellipodia and filopodia under the endothelium in order to transmigrate. Rho GTPases play key roles in cell migration by regulating cytoskeletal dynamics and cell adhesion. We have found that the Rho GTPase RhoA is required for efficient T-cell polarization and migration on endothelial cells as well as transendothelial migration. RhoA-depleted cells lack both lamellipodia and uropods, and instead have narrow protrusions extending from a rounded cell body. Using a RhoA activity biosensor, we have shown that RhoA is active at the leading edge in lamellipodia and filopodia of crawling and transmigrating T cells, as well as in the uropod. In lamellipodia, its activity correlates with both protrusion and retraction. We predict that RhoA signals via the formin mDIA 1 during lamellipodial protrusion whereas it induces lamellipodial retraction via the kinase ROCK and actomyosin contractility. We propose that different guanine-nucleotide exchange factors (GEFs) are responsible for coordinating RhoA activation and signaling in different regions of transmigrating T cells.