Isothiocyanates are sulfur-containing compounds which are largely responsible for the typical flavor of cruciferous vegetables. In animals and humans they are conjugated with glutathione; the first product of this reaction is a dithiocarbamate, which can be ultimately metabolized to the corresponding mercapturic acid, excreted in urine. Aliphatic and aromatic isothiocyanates, as well as dithiocarbamates, can react quantitatively with an excess of 1,2-benzenedithiol giving rise to a cyclic condensation product readily measured in biological fluids. The use of this assay as a biomarker of isothiocyanate or cruciferous vegetable intake can offer several advantages over food frequency questionnaires, mainly in the objectivity and accuracy of the measure and the lack of bias. Epidemiological studies on the relationship between cancer risk with isothiocyanate intake or excretion have shown inverse associations, mainly with lung cancer. The studies are also consistent in reporting a gene-environment interaction, with a stronger protective effect in persons null for the GSTM1 or GSTT1 genotype. However, further research is needed to better clarify the importance of these polymorphisms in modulating the effect of isothiocyanates in cancer development.