A polymerase chain reaction (PCR) method was developed that was capable of detecting a wide range of medically important fungi from clinical specimens. The primer pair was designed in conserved sequences of 18S-ribosomal RNA genes shared by most fungi. The lower limit of detection of this PCR technique was 1 pg of Candida albicans genomic DNA by ethidium bromide staining and 100 fg after Southern analysis. A 687-bp product was amplified successfully by PCR from all 78 strains of 25 medically important fungal species studies, including Candida spp., Hansenula spp., Saccharomyces cerevisiae, Cryptococcus neoformans, Trichosporon beigelii, Malassezia furfur, Pneumocystis carinii, Aspergillus spp., and Penicillium spp., but not from any strains of Mucor spp., Escherichia coli, or methicillin-resistant Staphylococcus aureus (MRSA), calf thymus or human placenta. This specificity was subsequently confirmed by Southern analysis. PCR analysis of blood specimens collected from mice systemically infected with C. albicans and clinical samples including blood, cerebrospinal fluid and sputum appeared to be a more sensitive diagnostic method for invasive fungal infections than a conventional blood culture technique.