We previously demonstrated that a mAb to human neutrophils, designated 3H9, which was established by screening for inhibition of neutrophil adherence to plastic plates containing fetal bovine serum, enhanced both n-formyl-methionyl-leucyl-phenylalanine (FMLP)-induced chemotaxis and random migration of neutrophils. In the present study, we examined the mechanisms of 3H9-induced enhancement of neutrophil locomotion in the phagokinetic track assay. 3H9-induced neutrophil locomotion maintained a straight path which was different from the track resulting from FMLP-stimulated locomotion. This 3H9-induced migration required extracellular Ca2+. beta 2-Integrin activation was a prerequisite for the increase in cytosolic free calcium induced by 3H9 treatment. However, stimulation by 3H9 did not induce an increase in F-actin, even after CD18 activation. Signal transduction after molecular recognition by 3H9 was not mediated by pertussis toxin-sensitive, heterotrimeric G proteins. These results suggest that 3H9 enhances neutrophil migration by mechanisms which are different from those involved in usual chemoattractant-induced migration. Neutrophil surface molecules recognized by 3H9 may play a crucial role in the regulation of transendothelial migration of leukocytes.