Quantitative culture, quantitative PCR and the galactomannan enzyme immunoassay (EIA) were compared for their ability to determine the pulmonary fungal burden in a murine model of invasive aspergillosis. Quantitative culture of specimens containing hyphae under-represented the absolute fungal burden in established infection when compared with the two other methods. The best correlation was observed between the two non-culture methods. Higher variability was observed with the galactomannan EIA when compared with quantitative PCR. Collectively, these data suggest that quantitative PCR is the preferred method for determination of the pulmonary fungal burden in experimental aspergillosis.