Trials in free-living populations involving increased consumption of fruit and vegetables are difficult to monitor. We evaluated biomarkers for assessing fruit and vegetable intake and compliance in a 2-year trial. Postmenopausal women were randomised to 300 g additional fruit and vegetables per d (n 66), placebo (n 70) or potassium citrate (n 140). They completed dietary checklists (3-monthly) and food diaries or FFQ (yearly). We measured whole-blood folate, plasma vitamin C and homocysteine (yearly), serum vitamin E and carotenoids (at 12 months) and urinary vitamin K metabolites (yearly). Plasma vitamin C was associated with fruit and vegetable intake at baseline (r +0·31; P < 0·01), remaining significant only for the non-fruit and vegetable group at 12 months (r +0·43; P < 0·01). For the fruit and vegetable group, vitamin C increased by 5·9 μmol/l (P = 0·07) but was not significantly associated with fruit and vegetable intake; vitamin E, β-carotene and β-cryptoxanthin were higher compared with the non-fruit and vegetable group (P < 0·05); and whole-blood folate and the urinary 5C-aglycone metabolite of vitamin K were associated with vegetable intake. For all participants plasma vitamin C increased with increasing fruit and vegetable intakes, reaching a plateau of 90–95 μmol/l at intakes>500 g/d, whereas whole-blood folate, β-carotene and β-cryptoxanthin continued to increase. Concentrations of vitamin C, folate and β-cryptoxanthin were lower and the 7C-aglycone metabolite of vitamin K higher, in smokers compared with non-smokers. Suitable markers for monitoring fruit and vegetable compliance include β-carotene and β-cryptoxanthin. Plasma vitamin C and whole-blood folate may be suitable for monitoring intakes in populations but for monitoring compliance the former may be restricted to low intakes of fruit and vegetables and the latter to vegetable intake.