When macrophages phagocytose chitin (N-acetyl-D-glucosamine polymer) microparticles, MAPK are immediately activated, followed by the release of Th1 cytokines, but not IL-10. To determine whether phagocytosis and macrophage activation in response to chitin microparticles are dependent on membrane cholesterol, RAW264.7 macrophages were treated with methyl--cytodextrin (MBCD) and stimulated with chitin. These results were compared to the corresponding effects of bacterial components including heat-killed (HK) Mycobacterium bovis BCG (BCG) and an oligodeoxynucleotide of bacterial DNA (CpG-ODN). The MBCD treatment did not alter chitin binding or the phagocytosis of chitin particles 20 min after stimulation. At the same time, however, chitin-induced phosphorylation of cellular MAPK was accelerated and enhanced in an MBCD dose dependent manner. The increased phosphorylation was also observed for chitin phagosome-associated p38 and ERK1/2. In contrast, CpG-ODN and HK-BCG induced activation of MAPK in MBCD-treated cells at levels comparable to, or only slightly more than, those of control cells. We also found that MBCD treatment enhanced the production of TNF- and the expression of cyclooxygenase 2 (COX-2) in response to chitin microparticles. In neither MBCD- nor saline- treated macrophages, did chitin particles induce detectable IL-10 mRNA synthesis. CpG-ODN-induced TNF- production, and COX-2 expression were less sensitive to MBCD treatment. Among the agonists studied, our results indicate that macrophage activation by chitin microparticles was most sensitive to cholesterol depletion, suggesting that membrane structures integrated by cholesterol are important for physiologic regulation of chitin microparticle-induced cellular activation.