Microbiologic cultures of fungi are routinely incubated at ambient temperatures in room air, and the rate of recovery of Aspergillus species from clinical specimens is poor. Failure of current culture methods to mimic the physiologic temperature and low-oxygen environment found in hypha-laden infected tissue may underlie this poor recovery. Experiments were performed to compare the recovery of Aspergillus spp. incubated at 35 degrees C in 6% O(2)-10% CO(2) with that at 25 degrees C in room air. The samples tested included Aspergillus-infected tissue specimens from a dog model and human autopsies, experimental anaerobically stressed Aspergillus inocula, and 10,062 consecutive clinical specimens. Culture at 35 degrees C in 6% O(2)-10% CO(2) significantly enhanced the recovery of Aspergillus spp. from the infected autopsy tissue samples. Incubation at 35 degrees C alone resulted in approximately 10-fold-improved culture recovery from the experimentally stressed hyphae, and the 6% O(2)-10% CO(2) atmosphere independently favored growth under temperature-matched conditions. Finally, incubation at 35 degrees C (in room air) improved the overall recovery of Aspergillus spp. from clinical specimens by 31%. Culture at 35 degrees C in a microaerobic atmosphere significantly enhances the recovery of Aspergillus spp. from various sources. Aspergillus hyphae growing in infected tissue appear to be adapted to the physiologic temperature and hypoxic milieu.