Deletion mutants of pseudorabies virus unable to express glycoprotein gIII, gI, or gp63 or double and triple mutants defective in these glycoproteins were constructed, and their virulence for day-old chickens inoculated intracerebrally was determined. Mutants of wild-type pseudorabies virus defective in glycoprotein gIII, gI, or gp63 were only slightly less virulent (at most, fivefold) for chickens than was the wild-type virus. However, mutants defective in both gIII and gI or gIII and gp63 were avirulent for chickens, despite their ability to grow in cell culture in vitro to about the same extent as mutants defective in gIII alone (which were virulent). These results show that gIII plays a role in virulence and does so in conjunction with gI or gp63. The effect of gIII on virulence was also shown when the resident gIII gene of variants of the Bartha vaccine strain (which codes for gIIIB) was replaced with a gIII gene derived from a virulent wild-type strain (which codes for gIIIKa); gIIIKa significantly enhanced the virulence of a variant of the Bartha strain to which partial virulence had been previously restored by marker rescue. Our results show that viral functions that play a role in the virulence of the virus (as measured by intracerebral inoculation of chickens) may act synergistically to affect the expression of virulence and that the ability of the virus to grow in cell culture is not necessarily correlated with virulence.