The D816V mutation of c-kit has been detected in patients with mastocytosis. This mutation leads to constitutive tyrosine kinase activation of Kit. Because stem cell factor (SCF), the ligand for Kit (CD117⁺), is a chemoattractant for CD117⁺ cells and one feature of mastocytosis is an abnormal collection of mast cells in tissues derived from CD34⁺CD117⁺ mast cell precursors, the hypothesis was considered that the D816V mutation would enhance chemotaxis of these precursor cells. Constructs encoding wild-type Kit or Kit bearing the D816V mutation were transfected into Jurkat cells, labeled with Calcein-am, and migration to SCF assessed in the presence or absence of tyrosine kinase inhibitors. Chemotaxis to SCF was enhanced in D816V transfectants compared to wild-type Kit transfectants (P < .002). Migration of both transfectants was inhibited by tyrosine kinase inhibitors, although D816V transfectants were more sensitive. Chemotaxis was next performed on CD34⁺CD117⁺ circulating mast cell precursors obtained from patients with mastocytosis. Analysis of prechemotaxis and migrated cells showed that whereas less than 10% in the prechemotaxis sample had the D816V mutation, 40% to 80% of migrated cells had this mutation. These results demonstrate that the D816V Kit mutation enhances chemotaxis of CD117⁺ cells, offering one explanation for increased mast cells observed in tissues of patients with mastocytosis.