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A possibility of using cycloheximide tolerance and/or sensitivity as an additional diagnostic tool for distinguishing morphologically related species within common small-spored Alternaria has been tested during this study.A total of 33 strains from four Alternaria species-groups, namely Alternaria alternata, Alternaria arborescens, Alternaria infectoria and Alternaria tenuissima were tested for their growth response to 100 μg m−1 cycloheximide in potato carrot agar. All A. infectoria strains were completely inhibited, showing no growth at all even after prolonged incubation. In contrast, all other strains representing the remnant three species exhibited a high resistance to this antibiotic.Cycloheximide sensitivity represents a further important physiological character for distinguishing A. infectoria from the three similar species.The relevance of these findings corresponds with the potential ability of the Alternaria species produce mycotoxins. Cycloheximide may be in future used in the design of selective media for the isolation of some potentially toxigenic food-borne Alternaria species such as A. alternata, A. tenuissima and/or A. arborescens, for example in screening cereals for toxigenic Alternaria spp. and for their direct separation from nontoxigenic representatives of A. infectoria species-group.