The early development of indigenous and introduced Scots pine (Pinus sylvestris L.) ectomycorrhiza in natural forest humus was examined using molecular fingerprinting techniques. Non-mycorrhizal or mycorrhizal (Suillus bovinus (L. ex Fr.) O. Kuntze or Paxillus involutus (Batsch ex Fr.) Fr.) seedlings were placed in transparent two-dimensional microcosms on a thin layer of sieved humus to allow observation of ectomycorrhizal development and fungal growth. Twelve ectomycorrhizal morphotypes, mainly based on colour, gross morphology and outer mantle structure, were identified over a 3-month period. No successional trends in mycorrhiza formation were observed. For further characterization, individual ectomycorrhiza representing the different morphotypes were subjected to analyses of esterase (EST) isozyme profiles and RFLPs of the internal transcribed spacer (ITS) from fungal rRNA genes following amplification using PCR. All 10 morphotypes analysed displayed different esterase isozyme profiles, and the characteristic S. bovinus species diagnostic band (S. b. EST8) was detected in two white morphotypes. Ten Pink and Black ectomycorrhiza were all separated into one and three groups, respectively. A fast-running plant-specific polymorphic locus (Z) was also confirmed in most ectomycorrhizal morphotypes. Diagnostic species-specific (S. bovinus and Pink) EST bands were detected in intact external mycelium colonizing soil. Successful amplification of the ITS from individual ectomycorrhiza, of eight different morphotypes, was found to be mainly influenced by the DNA template concentration. Different RFLPs (Hinf I, Mbo I and Hha I) of the ITS placed the white morphotypes into two groups, one corresponding to S. bovinus, in agreement with the esterase fingerprinting. By contrast, Pink and Beige morphotypes displayed different EST profiles but nearly identical RFLP fingerprints. The ITS amplification success rate of standardized DNA template concentrations from 10 individual Pink and Black ectomycorrhiza was 90 and 50%, respectively. In all successful amplifications, the black morphotype yielded the smallest ITS fragment, similar to earlier reports for Cenococcum geophilum, that gave identical RFLPs. The ecological significance of the observed mycorrhizal diversity and combined application of these two identification methods is discussed.