Fusarium head blight (FHB), caused by the fungus Fusarium graminearum, is associated with grain contamination with mycotoxins such as deoxynivalenol (DON) and zearalenone (ZEA). Unlike DON, less is known about factors affecting ZEA production during FHB epidemics. The objective of this study was to quantify ZEA contamination of wheat grain as influenced by temperature, relative humidity, FHB index (IND), grain maturation, simulated late-season rainfall, and harvest timing. Mean ZEA concentrations were low (<1.1 ppm) during the early stages of grain development (25 to 31 days after anthesis [DAA]) but rapidly increased 35 to 51 DAA in field experiments, particularly under rainy conditions. Five or ten consecutive days with simulated rainfall shortly before harvest greatly increased ZEA contamination. Similarly, extremely high levels of ZEA (51.8 to 468.6 ppm) were observed in grain from spikes exposed to 100% relative humidity (RH) at all tested temperatures and mean IND levels under controlled conditions. Interestingly, at RH <= 90%, ZEA concentrations were very low (0.1 to 3.6 ppm) at all tested temperatures, even at IND above 90%. At 100% RH, mean ZEA contamination was significantly higher at 20 and 25 degrees C (235.1 and 278.2 ppm) than at 30 degrees C (104.7 ppm). Grain harvested early and not exposed to rainfall had lower mean ZEA than grain harvested late and/or subjected to preharvest rainfall. This study was the first to associate ZEA contamination of grain from FHB-affected wheat spikes with temperature and moisture and show through designed experiments that early harvest could be a useful strategy for reducing ZEA contamination.