The detection of fungal pathogens in clinical samples by PCR requires the use of extraction methods that efficiently lyse fungal cells and recover DNA suitable for amplification. We used quantitative PCR assays to measure the recovery of DNA from two important fungal pathogens subjected to six DNA extraction methods. Aspergillus fumigatus conidia or Candida albicans yeast cells were added to bronchoalveolar lavage fluid and subjected to DNA extraction in order to assess the recovery of DNA from a defined number of fungal propagules. In order to simulate hyphal growth in tissue, Aspergillus fumigatus conidia were allowed to form mycelia in tissue culture media and then harvested for DNA extraction. Differences among the DNA yields from the six extraction methods were highly significant (P < 0.0001) in each of the three experimental systems. An extraction method based on enzymatic lysis of fungal cell walls (yeast cell lysis plus the use of GNOME kits) produced high levels of fungal DNA with Candida albicans but low levels of fungal DNA with Aspergillus fumigatus conidia or hyphae. Extraction methods employing mechanical agitation with beads produced the highest yields with Aspergillus hyphae. The MasterPure yeast method produced high levels of DNA from C. albicans but only moderate yields from A. fumigatus. A reagent from one extraction method was contaminated with fungal DNA, including DNA from Aspergillus and Candida species. In conclusion, the six extraction methods produce markedly differing yields of fungal DNA and thus can significantly affect the results of fungal PCR assays. No single extraction method was optimal for all organisms.Noncultivation methods are increasingly being used to overcome the poor diagnostic sensitivities and long turnaround times associated with the detection and identification of fungal pathogens in clinical samples by cultivation. One of these cultivation-independent methods is real-time PCR, which can rapidly detect and quantify fungal nucleic acid sequences in human tissue samples. Real-time quantitative PCR (qPCR) assays have detection thresholds that approach single-molecule sensitivity, and thus, little additional assay sensitivity can be achieved in the PCR itself by techniques such as targeting genes with multiple copies per fungal genome or increasing the total amount of DNA tested. The ultimate sensitivity of any PCR assay for the detection of fungal pathogens depends on the efficient lysis of fungal cells in the tissue sample and the purification of DNA that is free of PCR inhibitors. Fungi have cell walls that impede lysis and the recovery of nucleic acids. Few studies have focused on the critical DNA extraction stage of sample processing, in contrast to the multitude of studies on fungal PCR assay methods. Furthermore, highly sensitive and specific nucleic acid-based methods for the detection of fungi necessitate the use of DNA extraction reagents that are free of contaminating fungal nucleic acids.We sought to compare DNA extraction methods by using qPCR to measure th...