Sphingosine 1-phosphate (S1P) initiates T and B cell exit from lymphoid tissues by activating the S1P 1 receptor on lymphocytes. To define the mechanistic details of this ligand–receptor interaction, the biological activity of the S1P-blocking Ab Sphingomab was investigated. Treatment of mice with Sphingomab resulted in blood B and T cell lymphopenia. Although Sphingomab blocked S1P 1-mediated calcium flux and receptor downregulation by S1P in vitro, plasma from Sphingomab-treated mice demonstrated a 4-fold increase in S1P concentration and largely retained its stimulating activity on S1P receptors. Plasma-borne S1P was obviously not sufficiently inactivated by Sphingomab to account for the observed lymphopenia. Therefore, we addressed the local S1P-blocking activity of Sphingomab in spleen and peripheral lymph nodes (pLNs) as a potential cause of PBL depletion. Transwell chemotaxis assays revealed the migration of freshly isolated splenocytes, but not pLN cells to S1P. However, chemotaxis of pLN cells was regained after culture in S1P-low medium, and pLN cells isolated from Sphingomab-treated mice also revealed enhanced chemotaxis to S1P, indicating substantial local inactivation of S1P in pLN after Sphingomab treatment. We conclude that treatment with the S1P-blocking Ab Sphingomab induces lymphopenia by inactivating S1P locally in pLN and not systemically in plasma. Consequently, the presence of local S1P amounts in secondary lymphoid organs contributes to B and T cell egress.