The potential of high-performance liquid chromatography (HPLC) with diode-array detection for the profiling of mycotoxins in food samples has been demonstrated. A gradient elution reversed-phase chromatographic method coupled with a suitable extraction procedure was devised for the separation and detection of major Alternaria mycotoxins in foodstuffs. Altenuene, alternariol, alternariol methyl ether (dibenzo-alpha-pyrone derivatives), altertoxin-I and altertoxin-II (perylene derivatives) were profiled in extracts of artificially infected maize, rice and tomato samples and naturally contaminated sunflower seeds. First evidence of the occurrence of a new dibenzo-alpha-pyrone derivative in Alternaria cultures is also reported.