Epidemiological studies have linked consumption of broccoli to a reduced risk of colon cancer in individuals with the glutathione S-transferase M1 (GSTM1) null genotype. GSTs are involved in excretion and elimination of isothiocyanates (ITCs), which are major constituents of broccoli and other cruciferous vegetables and have cancer chemopreventive potential, so it is speculated that ITCs may play a role in protection against human colon cancer. However, there is a lack of data from animal studies to support this. We carried out a bioassay to examine whether sulforaphane (SFN) and phenethyl isothiocyanate (PEITC), major ITCs in broccoli and watercress, respectively, and their corresponding N:-acetylcysteine (NAC) conjugates, show any chemopreventive activity towards azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF) in F344 rats. Groups of six male F344 rats were treated with AOM subcutaneously (15 mg/kg body wt) once weekly for 2 weeks. SFN and PEITC and their NAC conjugates were administered by gavage either three times weekly for 8 weeks (5 and 20 micromol, respectively) after AOM dosing (post-initiation stage) or once daily for 3 days (20 and 50 micromol, respectively) before AOM treatment (initiation stage). The bioassay was terminated on week 10 after the second AOM dosing and ACF were quantified. SFN, SFN-NAC, PEITC and PEITC-NAC all significantly reduced the formation of total ACF from 153 to 100-116 (P < 0.01) and multicrypt foci from 52 to 27-38 (more than four crypts/focus; P < 0.05) during the post-initiation treatment. However, only SFN and PEITC were effective during the initiation phase, reducing the total ACF from 153 to 109-115 (P < 0.01) and multicrypt foci from 52 to 35 (more than four crypts/focus; P < 0.05). The NAC conjugates were inactive as anti-initiators against AOM-induced ACF. These findings provide important laboratory evidence for a potential role of SFN and PEITC in the protection against colon cancer.