A high intake of glucosinolate-containing cruciferous vegetables, such as Brussels sprouts (Brassica oleraceae), has been linked to a decreased cancer risk, but the underlying mechanism is still unclear. The aim of this study was to reveal possible modulating effects of consumption of Brussels sprouts on duodenal, rectal and lymphocytic (i) glutathione S-transferase (GST) enzyme activity, (ii) GST isozyme levels and (iii) glutathione (GSH) content. Ten healthy non-smoking volunteers were randomly assigned to two groups in a cross-over design. Five persons started on a glucosinolate-free diet (control period), while the other five consumed 300 g/day cooked Brussels sprouts, at the expense of 300 g glucosinolate-free vegetables (sprouts period). After 7 days the regimen was changed for a further week. At the end of both periods blood samples and duodenal and rectal biopsies were taken. Mean GST activity showed marked differences between duodenal, rectal and lymphocytic cytosols (737 ± 54, 321 ± 29 and 154 ± 14 nmol/min/mg protein respectively), but was uninfluenced by the dietary regimen. Isozyme distribution varied greatly between the tissues. In duodenum GST-α, -π and -μ isozymes were expressed in considerable amounts (8441 ± 1365, 3002 ± 223 and 536 ± 248 ng/mg protein respectively). Rectal biopsies also contained above three GST classes, but here GST-π was the most pronounced expressed isozyme (2849 ± 246) followed by GST-p (495 ± 242), while GST-α was only present in minor quantities (149 ± 31). In lymphocytes only GST-π (755 ± 96) and GST-μ (83 ± 54) could be detected. As a result of the dietary regimen rectal GST-α and -π levels were slightly increased at the end of the sprouts period, by 30 and 15% respectively. GSH contents were uninfluenced by the dietary regimen. In conclusion, consumption of glucosinolate-containing Brussels sprouts for 1 week results in increased rectal GST-α and -π isozyme levels. We hypothesize that these enhanced detoxification enzyme levels may partly explain the epidemiological association between a high intake of glucosinolates (cruciferous vegetables) and a decreased risk of colorectal cancer.