3-Acetyl deoxynivalenol (3-ADON) and esterase production were determined in a strain ofFusarium culmorum insensitive to the fungicide, difenoconazole. Following further exposure to this fungicide for different periods of time, the initiation of 3-ADON production was observed to be accelerated in the insensitive strain compared to a control (sensitive) strain of the phytopathogen. In particular, 3-ADON appeared in insensitive cultures within 21 days of incubation with fungicide levels of 1 to 4 µg/ml media and at 43 days with difenoconazole levels of 6 and 10 µg/ml. However, in the control strain, 3-ADON production was delayed until 28 and 57 days for the respective doses of fungicide. For these times and fungicide levels, the overall production of 3-ADON by the insensitive strain was significantly different from the zero values recorded with the control strain (P<0.05). Moreover, although difenoconazole was generally effective in depressing 3-ADON synthesis in both strains, the suppression was not significant (P>0.05) for the insensitive strain at 43 and 57 days of incubation with fungicide added at 1 µg/ml. In a parallel experiment, total esterase production was observed to increase progressively over time and at consistently higher levels for the insensitive strain from day 14 onwards so that by day 35 the strain difference was significant (P<0.05). Although the increase with time occurred in both strains, this enhancement appeared at 14 days in the insensitive strain (P<0.05) but delayed until 21 days in the control strain (P<0.05). In conclusion, these investigations have demonstrated the persistence of 3-ADON production and enhanced levels of total esterases in a strain ofF. culmorum insensitive to difenoconazole. Furthermore, it is proposed that changes in esterase profiles might be of diagnostic value in identifying toxigenic strains of differentFusarium species.