Non-culture methods being developed and evaluated for mycotic infections include polymerase chain reaction (PCR), galactomannan (GM) antigenemia, Western blot (WB) to detect antibodies, and detection of the fungal metabolites D-arabinitol and (1,3)-b-D-glucan. Sample preparation for PCR from blood specimens depends on fractionation of peripheral blood, its pre-incubation in blood culture broth, or a total DNA method, which does not rely on fractionation, or pre-incubation. Targets for PCR of fungi in the 18S or ITS2 subunits of the ribosomal RNA genes facilitated the design of Aspergillus and Candida genus and species probes. Amplicons were identi ed using PCR-enzyme linked immunosorbent assay (ELISA) or reverse lineblot formats. A pilot study indicated that PCR tests on blood specimens were positive at least once in patients with con rmed invasive aspergillosis (IA). When serum-PCR and serum-GM tests were compared in IA patients, antigenemia was more often positive. PCR detected Aspergillus DNA in bronchoalveolar lavage specimens from patients at risk even when cultures were negative. D-Arabinitol can be detected as a marker of candidiasis with gas chromatography-mass spectrometry or enzyme dependent- uorometry. Each method can differentiate the microbial D- and host L-enantiomers. (1,3)-b-D-Glucan is produced by most genera of pathogenic fungi and can be detected in plasma by the ‘G-test’. In patients with febrile neutropenia the ef cacy of azole therapy correlated with plasma (1,3)-b-D-glucan concentrations of ]10 pg ml¼1. The diagnosis of early acute pulmonary histoplasmosis can be improved by a WB test utilizing deglycosylated M antigen, a 94-kDa glycoprotein. The identity of M antigen as a catalase was deduced from the sequence of the cloned gene. PCR identi cation of Histoplasma capsulatum cultures was accomplished with primer pairs selected from H and M antigen gene sequences.