We tested the genotoxicity of nivalenol (NIV), a potent toxic trichothecene from Fusarium nivale, in cultured CHO cells and in several mouse organs and tissues (liver, kidney, thymus, bone marrow and mucosa of stomach, jejunum, and colon) using the alkaline single-cell gel electrophoresis (SCG, or Comet) assay. NIV at 50 and 100 micrograms/ml damaged the nuclear DNA of CHO cells in the absence of S9 mix, showing that NIV was a direct mutagen. In an in vivo study, mice were sacrificed 2, 4, and 8 h after either oral (20 mg/kg) or intraperitoneal (3.7 mg/kg) administration of NIV. DNA damage was measured by the SCG assay as modified by us. After oral dosing, DNA damage appeared in the kidney and bone marrow at 2 h (returning to almost control level within the following 2 h), and in the stomach, jejunum, and colon at 2, 4, and 8 h, respectively. Liver and thymus DNA were not damaged. After intraperitoneal injection, no DNA damage appeared in any of the organs or tissues tested except for the colon, where extensive DNA damage was observed, as in the oral study, at 8 h. For histopathological examination, mice were sacrificed 2, 4, and 8 h after oral (20 mg/kg) administration of NIV. No necrotic changes were detected in any of the organs where NIV yielded statistically significant DNA damage. To measure the effect of NIV on transport activity in mice, 10 ml/kg (same volume as NIV treatments) of 1% brilliant blue FCF (BB) was administered orally. Thirty minutes later, the BB reached the colon, and simultaneous oral administration of NIV (20 mg/kg, dissolved in 10 ml BB solution) did not affect the dye transport rate. Thus, the strong yet delayed damage to colon DNA may follow from a systemic absorption rather than a topical effect. As a direct mutagen, NIV showed organ specific genotoxicity in mice in time and intensity.