Here we report the characterization of fungi from 10 accretion ice sections (3300-5100 y old) as well as two deep glacial ice sections that are close to the bottom of the glacier (1,000,000-2,000,000 y old) from the Vostok, Antarctica, 5G ice core. Fungi were characterized by fluorescence microscopy culturing and sequence analyses of ribosomal DNA internal transcribed spacers. A total of 270 fungal colonies were cultured from the accretion ice of subglacial Lake Vostok and an additional 14 from the glacial ice immediately above the accretion ice. Mean concentrations were 0-4.42 cells mL(-1) ice meltwater of which 0-100% exhibited viability (as determined by fluorescence microscopy). Thirty-one unique fungal ribosomal DNA sequences (28 from accretion ice and three from glacial ice) were determined and compared to recent taxa. The results, plus tests for growth at low temperatures, indicated that Lake Vostok contains a mixture of heterotrophic psychrotolerant fungal species. This indicates that the lake is not sterile but contains a unique ecosystem.