CD13, a receptor for human coronavirus 229E (HCoV-229E), was identified as a major component of the Triton X-100-resistant membrane microdomain in human fibroblasts. The incubation of living fibroblasts with an anti-CD13 antibody on ice gave punctate labeling that was evenly distributed on the cell surface, but raising the temperature to 37°C before fixation caused aggregation of the labeling. The aggregated labeling of CD13 colocalized with caveolin-1 in most cells. The HCoV-229E virus particle showed a binding and redistribution pattern that was similar to that caused by the anti-CD13 antibody: the virus bound to the cell evenly when incubated on ice but became colocalized with caveolin-1 at 37°C; importantly, the virus also caused sequestration of CD13 to the caveolin-1-positive area. Electron microscopy confirmed that HCoV-229E was localized near or at the orifice of caveolae after incubation at 37°C. The depletion of plasmalemmal cholesterol with methyl ␤-cyclodextrin significantly reduced the HCoV-229E redistribution and subsequent infection. A caveolin-1 knockdown by RNA interference also reduced the HCoV-229E infection considerably. The results indicate that HCoV-229E first binds to CD13 in the Triton X-100-resistant microdomain, then clusters CD13 by cross-linking, and thereby reaches the caveolar region before entering cells.Recent studies have revealed that the plasma membranes of cells contain microdomains with discrete molecular compositions. Rafts are sphingolipid-and cholesterol-rich membrane microdomains that are thought of as platforms for signal transduction (39, 40). Although there are still many controversies regarding how rafts exist in living cells, it is generally agreed that cholesterol is indispensable for their integrity and that the detergent-resistant membrane (DRM) fraction is the in vitro correlate of the raft. Because acyl chains of sphingolipids and glycosylphosphatidylinositol (GPI)-anchored proteins enriched in the DRM fraction are more highly saturated than those of glycerolipids in the bulk membrane, the raft domain is thought to show less fluidity than nonraft areas of the plasma membrane. However, it is difficult to capture rafts morphologically because their shape and size are likely to change dynamically (40).On the other hand, caveolae were first defined morphologically as invaginations of the plasma membrane (49). They are also susceptible to cholesterol depletion (31). Moreover, caveolin-1, -2, and -3, which were identified as major components of caveolae (31,35,44,47), are highly enriched in the DRM fraction (2,12,14,36). Several results suggest that many molecules are shared by rafts and caveolae but that at least several molecules that are enriched in the DRM fraction are not concentrated in caveolae (11). Thus, caveolae are not simply a stabilized form of rafts, but there should be a regulatory mechanism (as yet unknown) to control the molecular distribution between caveolae and rafts.It has been shown that cross-linked raft molecules, such as GPI-anchored proteins,...