The mechanism of poxvirus attachment to cells is poorly understood. We have identified a 32-kDa envelope protein of vaccinia virus which binds to the surface of cultured cells. This binding is specific and selective (J.-S. Maa, J. F. Rodriguez, and M. Esteban, J. Biol. Chem. 265:22174-22180, 1990; C. Lai, S. Gong, and M. Esteban, J. Virol. 65:499-504, 1991). In this investigation, we studied the effect of inactivating the 32-kDa gene (32K gene) on the biology of vaccinia virus. We show that inactivation of the 32K gene decreases by 80% the mortality of mice infected with 32K- vaccinia virus. This reduction in mortality correlates with diminished viral gene expression in target tissues. In highly polarized epithelial cells, viral gene expression of 32K- virus was reduced (50 to 60%) at both the apical and basolateral surfaces in comparison with a 32K+ virus. Restriction of virus gene expression in polarized cell surfaces occurs for both intracellular and extracellular forms of infectious 32K- vaccinia virus. The two infectious forms of vaccinia virus 32K+ infect polarized cells preferentially by the basolateral surface. Our findings provide evidence of the importance of the 32-kDa protein in viral pathogenesis.