Glucosinolate consumption from brassica vegetables has been implicated in reduction of cancer risk. The isothiocyanate breakdown products of glucosinolates appear to be particularly important as chemoprotective agents. Before consumption, brassica vegetables are generally cooked, causing the plant enzyme, myrosinase, to be denatured, influencing the profile of glucosinolate breakdown products produced. Some human intestinal microflora species show myrosinase-like activity (e.g. bifidobacteria). We aimed to increase bifidobacteria by offering a prebiotic (inulin) in a randomised crossover study. Six volunteers consumed inulin (10 g/d) for 21 d followed by a 21 d control period (no inulin). Treatment periods were reversed for the remaining six volunteers. During the last 5 d of each period two cabbage-containing meals were consumed. Total urine output was collected for 24 h following each meal. Cabbage was microwaved for 2 min (lightly cooked) or 5.5 min (fully cooked). Faecal samples were collected at the start and after the inulin and control treatments. Bifidobacteria were enumerated by real-time PCR. Allyl isothiocyanate production was quantified by measuring urinary excretion of allyl mercapturic acid (AMA). Bifidobacteria increased following prebiotic supplementation (P < 0.001) but there was no impact of this increase on AMA excretion. AMA excretion was greater following consumption of lightly cooked cabbage irrespective of prebiotic treatment (P < 0.001). In conclusion, the most effective way to increase isothiocyanate production may be to limit the length of time that brassica vegetables are cooked prior to consumption.