The migration of immunocytes within the extracellular matrix (ECM) is influenced by the activation state of the incoming cell and its responses to the presence of chemokines and cytokines. We studied the regulatory role of TGF‐β1 on T cell homing to secondary lymphatic organs, such as the spleen, and chemotaxis within an ECM‐like environment in using an ECM‐like 3‐dimensional gel system designed to follow the migration of individual leukocytes along chemokine gradients in real time. The numbers of migrating naive, but not memory T cells toward SDF‐1α markedly increased after pre‐incubating the cells with TGF‐β1 (0.25 ng / ml) for 24 h. The mechanisms underlying TGFβ1‐modulated migration involve the up‐regulation of the expression of the SDF‐1α receptor CXCR4, the enhancement of the SDF‐1α‐induced actin polymerization, and increased phosphorylation of Pyk2, a focal adhesion kinase involved in integrin‐mediated lymphocyte migration, adhesion and interactions with ECM. Interestingly, priming of naive human T cells with TGF‐β1 increased homing of these cells to the spleen of NOD / SCID mice in a CXCR4‐dependent manner. We propose that theeffect of TGF‐β1 on the chemotaxis of naive T cells may be important in the locomotion of naive T cells toward SDF‐1α‐rich niches.