In response to chemoattractants neutrophils extend an actin-rich pseudopod, which imparts morphological polarity and is required for migration. Even when stimulated by an isotropic bath of chemoattractant, neutrophils exhibit persistent polarization and continued lamellipod formation at the front, suggesting that the cells establish an internal polarity. In this report, we show that perturbing lipid organization by depleting plasma membrane cholesterol levels reversibly inhibits cell polarization and migration. Among other receptor-mediated responses, beta(2) integrin up-regulation was unaffected, and initial calcium mobilization was only partially reduced by cholesterol depletion, indicating that this treatment did not abrogate initial receptor-mediated signal transduction. Interestingly, cholesterol depletion did not prevent initial activation of the GTPase Rac or an initial burst of actin polymerization, but rather it inhibited prolonged activation of Rac and sustained actin polymerization. Collectively, these findings support a model in which the plasma membrane is organized into domains that aid in amplifying the chemoattractant gradient and maintaining cell polarization.