ABSTRACTEpstein-Barr virus (EBV) uses different virus and cell proteins to enter its two major targets, B lymphocytes and epithelial cells. The routes that the virus takes into the two cell types are also different. To determine if these differences extend to movement from the cell surface to the nucleus, we examined the fate of incoming virus. Essentially all virus that entered a B cell remained stable for at least 8 h. In contrast, up to 80% of virus entering an epithelial cell was degraded in a compartment sensitive to inhibitors of components involved in autophagy. Inhibitors of actin remodeling blocked entry into a B cell but had no effect or enhanced entry into an epithelial cell. Inhibitors of the microtubule network reduced intracellular transport in both cell types, but movement to the nucleus in an epithelial cell also required involvement of the actin cytoskeleton. Deletion of the cytoplasmic tail of CR2, which in an epithelial cell interacts with the actin nucleator FHOS/FHOD when cross-linked by EBV, had no effect on infection. However, inhibitors of downstream signaling by integrins reduced intracellular transport. Cooperation of the microtubule and actin cytoskeletons, possibly activated by interaction with integrin binding proteins in the envelope of EBV, is needed for successful infection of an epithelial cell.