We have developed a capillary tube assay in combination with real-time PCR to quantitate the number of chemoattracted Leptospira cells. We identified Tween 80, glucose, sucrose, and pyruvate as attractants for Leptospira cells; amino acids and vitamin B(12) were found to be nonchemotactic or weakly chemotactic. This assay has the general applicability to further our understanding of leptospiral chemotaxis.