We recently reported that disruption of the actin cytoskeleton enhanced N-formylmethionyl-leucyl-phenylalanine (fMLP)-stimulated granule exocytosis in human neutrophils, but decreased plasma membrane expression of complement receptor 1 (CR1), a marker of secretory vesicles. The present study was initiated to determine if reduced CR1 expression was due to fMLP-stimulated endocytosis, to determine the mechanism of this endocytosis, and to examine its impact on neutrophil functional responses. Stimulation of neutrophils with fMLP or ionomycin in the presence of latrunculin A resulted in uptake of AlexaFluor 488-labeled albumin and transferrin and reduced plasma membrane expression of CR1. These effects were prevented by preincubation of the cells with sucrose, chlorpromazine, or monodansylcadaverine (MDC), inhibitors of clathrin-mediated endocytosis. Sucrose, chlorpromazine and MDC also significantly inhibited fMLP- and ionomycin-stimulated specific and azurophil granule exocytosis. Disruption of microtubules with nocodazole inhibited endocytosis and azurophil granule exocytosis stimulated by fMLP in the presence of latrunculin A. Pharmacologic inhibition of phosphatidylinositol 3-kinase, extracellular signal-regulated kinase (ERK1/2), and protein kinase C significantly reduced fMLP-stimulated transferrin uptake in the presence of latrunculin A. Blocking clathrin-mediated endocytosis had no significant effect on fMLP-stimulated phosphorylation of ERK1/2 in neutrophils pre-treated with latrunculin A. From these data we conclude that the actin cytoskeleton functions to limit microtubule-dependent, clathrin-mediated endocytosis in stimulated human neutrophils. The limitation of clathrin-mediated endocytosis by actin regulates the extent of both specific and azurophilic granule exocytosis.