The effects of cholesterol-perturbing agents on the mobilization of calcium induced upon the stimulation of human neutrophils by chemotactic factors were tested. Methyl-␤-cyclodextrin and filipin did not alter the initial peak of calcium mobilization but shortened the duration of the calcium spike that followed the addition of fMet-Leu-Phe. These agents also inhibited the influx of Mn 2؉ induced by fMet-Leu-Phe or thapsigargin. Methyl-␤-cyclodextrin and filipin completely abrogated the

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... ion of calcium induced by 10 ؊10 M platelet-activating factor, which at this concentration depends to a major extent on an influx of calcium as well as the influx of calcium induced by 10 ؊7 M platelet-activating factor. On the other hand, methyl-␤-cyclodextrin and filipin enhanced the mobilization of calcium induced by ligation of Fc␥RIIA, an agonist that did not induce a detectable influx of calcium. Finally, methyl-␤-cyclodextrin and filipin enhanced the stimulation of the profile of tyrosine phosphorylation, the activity of phospholipase D (PLD), and the production of superoxide anions induced by fMet-Leu-Phe. These results suggest that the calcium channels utilized by chemotactic factors in human neutrophils are either located in cholesterol-rich regions of the plasma membrane, or that the mechanisms that lead to their opening depend on the integrity of these microdomains.