Vascular adhesion molecules regulate the migration of leukocytes from the blood into tissue during inflammation. Binding of leukocytes to vascular cell adhesion molecule-1 (VCAM-1) activates signals in endothelial cells, including Rac1 and calcium fluxes. These VCAM-1 signals are required for leukocyte transendothelial migration on VCAM-1. However, it has not been reported whether the cytoplasmic domain of VCAM-1 is necessary for these signals. Interestingly, the 19-amino acid sequence of the VCAM-1 cytoplasmic domain is 100% conserved among many mammalian species, suggesting an important functional role for the domain. To examine the function of the VCAM-1 cytoplasmic domain, we deleted the VCAM-1 cytoplasmic domain or mutated the cytoplasmic domain at amino acid N724, S728, Y729, S730, or S737. The cytoplasmic domain and S728, Y729, S730, or S737 were necessary for leukocyte transendothelial migration. S728 and Y729, but not S730 or S737, were necessary for VCAM-1 activation of calcium fluxes. In contrast, S730 and S737, but not S728 or Y729, were necessary for VCAM-1 activation of Rac1. These functional data are consistent with our computational model of the structure of the VCAM-1 cytoplasmic domain as an α-helix with S728 and Y729, and S730 and S737, on opposite sides of the α-helix. Together, these data indicate that S728 and Y729, and S730 and S737, are distinct functional sites that coordinate VCAM-1 activation of calcium fluxes and Rac1 during leukocyte transendothelial migration.