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Dominance of melanin-containing forms in micromycete communities is a good marker of unfavorable conditions in biotopes. On the other hand, according to the theory of humus formation through melanoides, fungal melanins can be involved in the synthesis of humus substances. Many species of micromycetes produce dark brown polymers, and thereby might contribute directly to the pool of humus substances creating a buffer organic matrix. It is known that communities in biotopes rich in humus substances demonstrate increased resistance to toxic anthropogenic stress. However, mechanisms of interaction of humus substances and fungal metabolites were little investigated. One of the advantageous tools to analyze the interaction of fungal metabolites and humus substances can serve spectral analysis, allowing using absorbance values and fluorescence characteristics to record the results of the mutual influence of extracellular fungal metabolic products and dissolved humus substances. The work objective was to study spectral features of chromophoric organic substances released by soil fungi in Chapek-medium without and in presence of humus substances. Spectral properties of fungal metabolites in liquid medium were measured and compared with that for natural humus substances and commercial humate preparations in water. Chromophoric organic matter released by various fungi strains showed absorption and fluorescence spectra similar in main spectral features for the samples with different pigmentation of mycelium: decreasing absorbance values towards longer wavelengths with a shoulder at 280-290 nm and fluorescence emission spectra with two overlapping bands (UV fluorescence of phenolics and proteins within 300-350 nm and blue fluorescence of fungal polymers). After two-three weeks of cultures growing in the aqueous humate solution its fluorescence characteristics became more similar to humic-type fluorescence of natural water. Wavelength of emission maximum and quantum yield of humic-type fluorescence were found to be excitation wavelength dependent because of increased heterogeneity of humus substances in culture medium compared to initial commercial humate solution. Transformations in humus substances composition caused by microscopic fungi have been monitored and characterized using spectral measurements.