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The ecology and stress adaptation of black rock inhabiting fungi in hot and cold extreme environments are not yet well understood. Two-dimensional gel electrophoresis (2-DE) is a promising tool to study the protein expression profiling and the metabolic status of microorganisms under stress conditions. The sample preparation has been shown to be the bottleneck for high resolution protein separation in 2-DE. For this purpose conditions must be optimized to obtain reliable and reproducible results. In addition, due to a multilayered and strongly melanized cell wall of black microcolonial fungi, special protocols for cell disruption and processing are required. In the present study, the protocol for protein extraction was established and optimized for the black yeast Exophiala jeanselmei MA 2853. The same protocol was successfully examined also for the meristematic fungus Coniosporium perforans MA 1299. Among the three procedures evaluated, trichloroacetic acid (TCA) precipitation, TCA/acetone precipitation, and phenol extraction combined with methanol/ammonium acetate precipitation, the latter showed to be the best method for black yeasts and meristematic fungi. Penicillium chrysogenum was used as reference strain. (C) 2011 British Mycological Society. Published by Elsevier Ltd. All rights reserved.