Aspergillus fumigatus is a clinically important fungus with the ability to cause invasive aspergillosis with high mortality rates in immunocompromised patients and chronic pulmonary aspergillosis in immunocompetent individuals. Virulence of mutants has traditionally been assessed using mammalian hosts such as mice and rats and more recently the fruit fly, Drosophila melanogaster, demonstrated the potential to act as an in vivo host suitable for screening Aspergillus mutants. In this study using a larger thermotolerant invertebrate, Galleria mellonella, the virulence of individual gene deletants of Aspergillus fumigatus (cpcA, sidA, sidC, sidD, sidF and paba,) were compared to the parental and gene-replacement strains, if available. A range of infectious challenges consisting of from 3 x 10(3)-3 x 10(6) spores/larva was followed by observation of larval survival with mean survival time used as a surrogate of microbial pathogenicity. Mutants cpcA, sidA, sidF and paba were avirulent and sidC and sidD showed attenuated virulence. Virulence assessment in G. mellonella correlated closely with the historic data generated using mice and Drosophila. Pre-screening Aspergillus mutants using G. mellonella could significantly reduce the number of mammals required to assess changes in virulence.