Vaccinia virus gene B5R encodes a M(r) 42K glycoprotein that is expressed throughout infection and forms part of the envelope of extracellular virus. In this paper deletion mutants (delta B5R) lacking the B5R open reading frame (ORF) from the Western Reserve (WR) and IHD-J strains of vaccinia virus have been constructed and shown to form very small plaques compared with the wild-type viruses. This phenotype was directly attributable to loss of the B5R gene since re-insertion of this gene from WR or IHD-J into the WR mutant lacking B5R (W-delta B5R) restored a normal plaque phenotype. In the latter case the failure of the revertant to form comets indicated that the nine amino acid differences in the B5R ORF between the IHD-J and WR strains of virus are not responsible for comet formation by IHD-J virus. Furthermore, the B5R deletion mutant of IHD-J (I-delta B5R) still formed small comets. Despite the small plaque phenotype of the deletion mutants, normal yields of intracellular naked virus (INV) were produced. In contrast, deletion of B5R had a profound affect on the formation of the extracellular enveloped virus (EEV). Transmission electron microscopy indicated that INV particles were not wrapped by a double layer of Golgi-derived membrane and enveloped particles were not detected within the cell or on the cell surface without expression of the B5R protein. Biochemical measurement of EEV formation, by labeling infected cells with [3H]thymidine followed by cesium chloride density gradient centrifugation of particles released from the cells 24 hr postinfection, showed that only 10% of WT levels of EEV were produced by I-delta B5R. The loss of the B5R ORF caused severe attenuation in intranasally infected mice. At doses between 10(4) and 3 x 10(7) plaque-forming units there were no signs of disease in animals infected with W-delta B5R, whereas at comparable doses the WR parent virus caused significant mortalities. Finally, an ORF with 93.4% amino acid identity to vaccinia WR B5R is present in variola major virus strain Harvey and the B5R protein was shown by Western blotting to be expressed by all orthopoxviruses tested.