The interaction between the integrin α 4 β 7 and its ligand, mucosal addressin cell adhesion molecule-1, on high endothelial venules represents a key adhesion event during lymphocyte homing to secondary lymphoid tissue. Stromal cell-derived factor-1α (SDF-1α) is a chemokine that attracts T and B lymphocytes and has been hypothesized to be involved in lymphocyte homing. In this work we show that α 4 β 7-mediated adhesion of CD4+ T lymphocytes and the RPMI 8866 cell line to mucosal addressin cell adhesion molecule-1 was up-regulated by SDF-1α in both static adhesion and cell detachment under shear stress assays. Both naive and memory phenotype CD4+ T cells were targets of SDF-1α-triggered increased adhesion. In addition, SDF-1α augmented α 4 β 7-dependent adhesion of RPMI 8866 cells to connecting segment-1 of fibronectin. While pertussis toxin totally blocked chemotaxis of CD4+ and RPMI 8866 cells to SDF-1α, enhanced α 4 β 7-dependent adhesion triggered by this chemokine was partially inhibited, indicating the participation of Gα i-dependent as well as Gα i-independent signaling. Accordingly, we show that SDF-1α induced a rapid and transient association between its receptor CXCR4 and Gα i, whereas association of pertussis toxin-insensitive Gα 13 with CXCR4 was slower and of a lesser extent. SDF-1α also activated the small GTPases RhoA and Rac1, and inhibition of RhoA activation reduced the up-regulation of α 4 β 7-mediated lymphocyte adhesion in response to SDF-1α, suggesting that activation of RhoA could play an important role in the enhanced adhesion. These data indicate that up-regulation by SDF-1α of lymphocyte adhesion mediated by α 4 β 7 could contribute to lymphocyte homing to secondary lymphoid tissues.