Contamination of Canadian barley samples by 3,15-diacetyldeoxynivalenol was detected by enzyme immunoassays combined with liquid chromatography-mass spectrometry. This is the first reported natural occurrence of this mycotoxin. The barley was infected mainly with Fusarium graminearum. Deoxynivalenol, 3-acetyldeoxynivalenol, and zearalenone were also found.