Previous experiments have established that late-season water deficit conditions and 28–30.5 C in the geocarposphere are optimum for preharvest aflatoxin contamination of Florunner peanuts. Visibly-undamaged peanuts from plants exposed to these stress conditions during the last 45–50 days before harvest were highly contaminated with aflatoxin at harvest. The objective of this study was to determine the duration of water and soil temperature stress required for extensive preharvest invasion by Aspergillus flavus and contamination of peanuts with aflatoxin. Stress conditions were imposed 20, 30, 40 and 50 days before harvest. Incidence of A. flavus was greatest in edible peanuts from the 30, 40, and 50 day stress treatments. A stress period of 20 days before harvest did not result in aflatoxin contamination of edible peanuts by A. flavus. Peanuts subjected to defined temperature and water deficit stress conditions for 30, 40, and 50 days became contaminated, therefore, a threshold stress period for preharvest aflatoxin contamination of peanuts by A. flavus was more than 20, and possibly less than 30 days before harvest.