Aims: To examine the effect of interactions between water, temperature and gas composition on growth and ochratoxin A (OTA) production by isolates of Penicillium verrucosum in vitro and in situ on grain-based media and wheat grain. Methods and Results: Three isolates of P. verrucosum were examined in relation to radial growth rate and OTA production, and to interacting conditions of water activity (a(w)), temperature and gas composition on a milled wheat medium. Subsequently, detailed temporal studies were carried out on gamma irradiated wheat grain over the range 0.75-0.995 a(w), 10-25 degrees C and air, 25 or 50% CO2. This showed that optimum growth of P. verrucosum was at 0.98 a(w) in vitro at 25 degrees C, but at 0.95 a(w) and 25 degrees C on wheat grain. The a(w) minimum for growth was about 0.80 a(w), although no OTA was produced under this condition even after 56 days. Significant inhibition of growth and OTA production occurred with 50% CO2, and 0.90-0.995 a(w) at 25 degrees C. Conclusions: The optimum and marginal conditions for growth and OTA production on wheat grain have been identified. At least 50% CO2 is needed to inhibit growth and OTA production by > 75% in moist grain (0.90-0.995 a(w)). Significance and Impact of the Study: First detailed identification of optimal and marginal interacting conditions of water/temperature and gas composition on growth and OTA production by P. verrucosum on wheat grain. This is a critical component of the postharvest management strategy for minimizing contamination by this important mycotoxin and predicting risk, based on environmental conditions, during drying and storage.