We have studied the effects of inhibiting the initial steps in processing of asparagine-linked oligosaccharides on the formation of vesicular stomatitis virus (VSV). Our data show that conditions which prevent the removal of glucose can block the growth of this virus. Our conclusion that inhibition of VSV synthesis is due specifically to an effect on the ability of the virus glycoprotein, G, to mature to a correct functional conformation is based on the following observations: (i) two drugs, deoxynojirimycin and castanospermine , both of which selectively inhibit the processing glucosidases, affected virus growth; (ii) only one of the two strains (San Juan and Orsay ) of VSV tested was affected and that strain, VSV(San Juan), is known to have a G protein highly sensitive to alterations in oligosaccharide structure; (iii) the effect was to make the formation of VSV(San Juan) temperature-sensitive, a result previously observed with alterations in the oligosaccharides on G protein; (iv) a cell variant missing glucosidase II activity also became temperature-sensitive in its ability to produce VSV(San Juan) but not VSV( Orsay ). Although inhibition of glucosidase activity by 1- deoxynojirimycin caused a 10-fold drop in virion formation, transport of G protein to the plasma membrane was not altered. The growth of VSV(San Juan) at 40 degrees C was not affected when subsequent steps in the processing pathway were blocked. These data indicate that by the time the glucose residues are removed G has attained a stable conformation.