Ribosomal DNA sequences provide new molecular approaches to characterise fungal species from the environment. This study presents a culture-independent approach to assessing fungal diversity by direct amplification, cloning, restriction digestion and sequencing of internal transcribed spacers (ITS). We have assessed this novel approach both with culturable soil fungi and with total environmental DNA extracted from the same soil sample. Sixty-seven colonies and 51 cloned amplicons from total DNA were compared on the basis of their ITS restriction patterns. Then, 58 representative ITS sequences were determined and classified by comparing their sequences with ITS sequences of known origin. All culturable fungi were ascomycetes with one basidiomycete exception. In contrast, cloned amplicons from total environmental DNA were identified to ascomycetes, one basidiomycete, one zygomycete and to several fungi belonging to the kingdoms Chromista and Protozoa. The results show that PCR–RFLP of ITS provides an efficient culture-independent molecular tool for ecological studies and to assess fungal diversity.