Using Fura-2AM microfluorimetry, we have shown for the first time that preincubation of macrophages with methyl-β-cyclodextrin, inducing cholesterol extraction from membranes and raft disruption, leads to significant inhibition of thapsigargin-induced store-dependent Ca2+ entry in rat peritoneal macrophages. In contrast, macrophage treatment with methyl-β-cyclodextrin after Ca2+ entry mechanisms were activated by store depletion by thapsigargin application leads to potentiation of subsequent store-dependent Ca2+ entry. The results suggest that intact lipid rafts are necessary for the activation but not the maintenance of store-dependent Ca2+ entry in macrophages.