ABSTRACT Gliotoxin is a nonribosomal peptide produced by Aspergillus fumigatus . This compound has been proposed as an A. fumigatus virulence factor due to its cytotoxic, genotoxic, and apoptotic properties. Recent identification of the gliotoxin gene cluster identified several genes ( gli genes) likely involved in gliotoxin production, including gliZ , encoding a putative Zn ₂ Cys ₆ binuclear transcription factor. Replacement of gliZ with a marker gene ( ΔgliZ ) resulted in no detectable gliotoxin production and loss of gene expression of other gli cluster genes. Placement of multiple copies of gliZ in the genome increased gliotoxin production. Using endpoint survival data, the ΔgliZ and a multiple-copy gliZ strain were not statistically different from the wild type in a murine pulmonary model; however, both the wild-type and the multiple-copy gliZ strain were more virulent than ΔlaeA (a mutant reduced in production of gliotoxin and other toxins). A flow-cytometric analysis of polymorphonuclear leukocytes (PMNs) exposed to supernatants from wild-type, ΔgliZ , complemented ΔgliZ , and ΔlaeA strains supported a role for gliotoxin in apoptotic but not necrotic PMN cell death. This may indicate that several secondary metabolites are involved in A. fumigatus virulence.