Abstract The objective of this study was to determine the effects of water activity ( a W; 0.900–0.995), temperature (5, 15, 25 and 30 °C), time of incubation (7–49 days) and their interactions on mycelial growth and deoxynivalenol (DON) production on irradiated wheat grain by two strains of Fusarium graminearum isolated from wheat ears in Argentina . Optimal a W levels for growth were in the range 0.950–0.995 with a temperature optima of 25 °C. Maximum growth rates were obtained at the highest a W (0.995) and 25 °C for both strains. No growth was observed at 5 °C regardless of the a W levels assayed. Both strains were able to growth at the lowest a W assayed (0.900), although the temperature ranges allowing growth at this minimal a W was 15–25 °C. DON was produced the most rapidly (7 days) when incubated at 25 °C and 0.995 a W. All other conditions required 7–14 days before DON was produced on grain. Maximum amounts of DON for both strains were produced at the highest a W treatment (0.995) after 6 weeks at 30 °C. The range of DON concentrations varied considerably (5 to 140,000 ng g − 1 ) depending on a W and temperature interaction treatments. Production of DON occurred over a narrower range of a W (0.995–0.95) than that for growth (0.995–0.90). DON was more rapidly produced at 25 °C but the maximum amount produced was at 30 °C. Two-dimensional profiles of a W × temperature were developed from these data to identify areas where conditions indicate a significant risk from DON accumulation.