The aim of this study was to evaluate the effects of postharvest temperature, light and UV-B irradiation on the flavonol content in broccoli using a multi-level design simulating commercial storage conditions from harvest until consumer purchase. The flavonol content in broccoli flower buds was examined during pre-storage and storage of broccoli heads, representing refrigerated transport with wholesale distribution and retail, respectively. Broccoli heads were pre-stored for four or seven days at 0 or 4°C in the dark and then stored for three days at 10 or 18°C. During storage, the broccoli heads were exposed for 12h per day to three different levels of visible light (13, 19 or 25μmolm−2s−1) or a combination of visible light (19μmolm−2s−1) and UV-B irradiation (0.23Wm−2), or they were stored in the dark. The contents of flavonols (quercetin, kaempferol and isorhamnetin), measured by HPLC as aglycons in flower bud extracts, were not significantly influenced by pre-storage period, pre-storage temperature or storage temperature. However, the interaction between pre-storage period and pre-storage temperature was found significant for flavonol contents during storage, with higher contents observed for broccoli pre-stored shorter (4d) at 4°C and longer (7d) at 0°C. The levels of epidermal flavonols, monitored with non-destructive repeated measurements of the broccoli heads during storage, were observed to increase with the duration of pre-storage and storage. General linear model analysis revealed that the radiation treatments significantly affected both quercetin content and epidermal flavonol levels in broccoli flower buds during storage, with the highest levels observed after a combination of visible light and UV-B irradiation treatment. However, the outcome of that postharvest treatment was influenced by a combination of factors including pre-storage period, pre-storage temperature and storage temperature. For one group of broccoli heads, that were pre-stored for four days at 4°C and exposed to visible light with UV-B irradiation during storage at 10°C, a significant 3.5-fold increase in the level of all flavonols was observed as compared with after pre-storage. The non-destructive measurements demonstrated that accumulation of epidermal flavonols upon UV-B exposure was initiated earlier in broccoli heads when stored at higher temperature (18°C).