Fusarium head blight (FHB) of wheat, caused by the fungus Fusarium graminearum, leads to grain contamination with mycotoxins such as deoxynivalenol (DON). Although FHB intensity is often positively correlated with DON, this relationship is quite variable and breaks down under certain conditions. One possible explanation for this could be the conversion of DON to DON-3-glucoside (D3G), a masked form of the toxin that is often missed by common DON testing methods. The objective of this study was to quantify the effects of temperature (20, 25, and 30^oC), relative humidity (RH, 70, 80, 90, and 100%), and pre-harvest rainfall patterns (continuous [Rain1], intermittent, or no supplemental rainfall [check]) on DON, D3G, and their relationship in grain from wheat spikes with different levels of FHB index (IND). D3G levels were higher in grain from spikes exposed to 100% RH than to 70, 80, or 90% RH at 20 and 25^oC across all tested IND levels. Mean D3G was highest at 20^oC. There were significant positive linear relationships between DON and D3G. Rainfall resulted in significantly higher mean D3G than the rain-free check. All rainfall treatments induced pre-harvest sprouting, as indicated by low falling numbers (FN), with Rain1 having the lowest mean FN. There were significant positive relationships between the rate of increase in D3G per unit increase in DON (a measure of conversion) and sprouting. As FN decreased, the rate of D3G conversion increased, and this rate of conversion per unit decrease in FN was greater at low than at high mean DON levels. These results provide strong evidence that moisture after FHB visual symptom development influenced DON-to-D3G conversion. To our knowledge, this study was the first to: 1) associate cooler, humid conditions with high levels of D3G contamination of wheat grain, 2) quantify associations among rainfall after visual FHB symptom development, D3G, DON, DON-to-D3G conversion, and pre-harvest sprouting across different baseline IND levels, and 3) model D3G contamination of grain as a function of DON and FN. These constitute valuable new information for understanding this complex disease-toxin system.