Daikon (Raphanus sativus L.), which is one of the cruciferous crops, has the largest production and consumption among the vegetables grown in Japan. Daikon quality such as flavor and color depends mainly on the contents of 4-methylthio-3-butenyl glucosinolate (MTB-GSL) and its breakdown products in both fresh and processed foods. The pungent flavor of daikon root is enzymatically converted from MTB-GSL to the corresponding volatile isothiocyanate (MTB-ITC) by myrosinase. MTB-ITC is known to possess antifungal and antibacterial activities against a range of organisms. The degradation product of MTB-ITC in water is likely to be the precursor of yellow pigment in daikon pickles. A quantitative method for determining MTB-GSL was developed by using a gas liquid chromatography after enzymatic hydrolysis of glucosinolates to elucidate the effect of environmental and genetic factors on it. Regarding the distribution of MTB-GSL content in a root, the phloem of tip was the highest with a gradual decline toward the stem and xylem. The change of MTB-GSL content after sowing was low at the early stage and rapidly reached a maximal peak followed by a gradual decline. There were great varietal differences in MTB-GSL contents among the 20 daikon cultivars tested with a coefficient of variation of 39%, and the contents in the 13 cultivars in two years were positively correlated. Application of sulfate to a nutrient solution caused a high accumulation of total glucosinolate in daikon roots.