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Although sphingosine-1-phosphate receptor 1 (S1P 1 ) has been shown to trigger several S1P targeted functions such as immune cell trafficking, cell proliferation, migration, or angiogenesis, tools that allow the accurate detection of endogenous S1P 1 localization and trafficking remain to be obtained and validated. In this study, we developed and characterized a novel monoclonal S1P 1 antibody. Mice were immunized with S1P 1 produced in the yeast Pichia pastoris and nine hybridoma clones producing monoclonal antibodies were created. Using different technical approaches including Western blot, immunoprecipitation and immunocytochemistry, we show that a selected clone, hereinafter referred to as 2B9, recognizes human and mouse S1P 1 in various cell lineages. The interaction between 2B9 and S1P 1 is specific over receptor subtypes, as the antibody does not binds to S1P 2 or S1P 5 receptors. Using cell-imaging methods, we demonstrate that 2B9 binds to an epitope located at the intracellular domain of S1P 1 ; reveals cytosolic and membrane localization of the endogenous S1P 1 ; and receptor internalization upon S1P or FTY720-P stimulation. Finally, loss of 2B9 signal upon knockdown of endogenous S1P 1 by specific small interference RNAs further confirms its specificity. 2B9 was also able to detect S1P 1 in human kidney and spinal cord tissue by immunohistochemistry. Altogether, our results suggest that 2B9 could be a useful tool to detect, quantify or localize low amounts of endogenous S1P 1 in various physiological and pathological processes.