Deep-seated fungal infection present with non specific symptoms and involve a large number of different organisms. DNA-based technology offers for eariler detection of fungal pathogens and then earlier initiation of antifungal therapy. In this study universal primers common to almost all fungi were used to amplification of internal transcribe spacer 1 and 2 region. Subsequent restriction enzyme analysis of PCR products, using HpaII allows us to identify the most medically opportunistic important fungi: Candida albicans, C. glabrata, C. tropicalis, C. kruzei, C. guilliermondi, Cryptococcus neoformans, Aspergillus fumigatus and Fusarium solani, according to sizely different bands in polyacrilamid gel electrophoresis. It seems that this panel of PCR-RFLP could be a rapid and useful molecular approach in diagnostic studies of invasive opportunistic fungal infections.