Copper is a critical metal of modern industry, and is the most widespread heavy metal contaminant in wastewater. Therefore, isolation of copper-tolerant microbes having the potential as biosorbent is fascinating not only from an environmental microbiology, but also from a biotechnology view point. In this study, we attempted to isolate highly copper-tolerant microbes from soil samples of the Nabanobori copper mine, the oldest mine in Japan. As a result of an enrichment culture, two fungal strains were isolated from soil of the smelter remains. The isolates could grow in a maximum of 200 mmol l(-l) Cu2+, and grew under a wide pH range. The Cu2+-binding capacity of nontreated biomass of the isolates was around 35 mg Cu2+ g(-1)-biomass. Analysis of 18S rDNA suggested that the isolates belong to the Aspergillus/Penicillium clade, but they represented a distinct lineage against known neighbours. The isolates were highly copper-tolerant, and their Cu2+-binding capacity was comparable to well-studied fungal sorbents. The isolates were implied as novel species. Soil of the historic old mine under weather-beaten conditions might be a suitable source for metal-tolerant microbes. The present results advance our understanding of metal-tolerant microbes, and offer a new tool for both environmental control and metal recovery operations.