Many viruses take advantage of receptor-mediated endocytosis in order to enter target cells. We have utilized influenza virus and Semliki Forest virus (SFV) to define a role for protein kinase C betaII (PKCbetaII) in endocytic trafficking. We show that specific PKC inhibitors prevent influenza virus infection, suggesting a role for classical isoforms of PKC. We also examined virus entry in cells overexpressing dominant-negative forms of PKCalpha and -beta. Cells expressing a phosphorylation-deficient form of PKCbetaII (T500V), but not an equivalent mutant form of PKCalpha, inhibited successful influenza virus entry-with the virus accumulating in late endosomes. SFV, however, believed to enter cells from the early endosome, was unaffected by PKCbetaII T500V expression. We also examined the trafficking of two cellular ligands, transferrin and epidermal growth factor (EGF). PKCbetaII T500V expression specifically blocked EGF receptor trafficking and degradation, without affecting transferrin receptor recycling. As with influenza virus, in PKCbetaII kinase-dead cells, EGF receptor was trapped in a late endosome compartment. Our findings suggest that PKCbetaII is an important regulator of a late endosomal sorting event needed for influenza virus entry and infection.