High intakes of fruits and vegetables, or high circulating levels of their biomarkers (carotenoids, vitamins C and E), have been associated with a relatively low incidence of cardiovascular disease, cataract and cancer. Exposure to a high fruit and vegetable diet increases antioxidant concentrations in blood and body tissues, and potentially protects against oxidative damage to cells and tissues. This paper describes blood concentrations of carotenoids, tocopherols, ascorbic acid and retinol in well-defined groups of healthy, non-smokers, aged 25-45 years, 175 men and 174 women from five European countries (France, UK (Northern Ireland), Republic of Ireland, The Netherlands and Spain). Analysis was centralised and performed within 18 months. Within-gender, vitamin C showed no significant differences between centres. Females in France, Republic of Ireland and Spain had significantly higher plasma vitamin C concentrations than their male counterparts. Serum retinol and alpha-tocopherol levels were similar between centres, but gamma-tocopherol showed a great variability being the lowest in Spain and France, and the highest in The Netherlands. The provitamin A: non-provitamin A carotenoid ratio was similar among countries, whereas the xanthophylls (lutein, zeaxanthin, beta-cryptoxanthin) to carotenes (alpha-carotene, beta-carotene, lycopene) ratio was double in southern (Spain) compared to the northern areas (Northern Ireland and Republic of Ireland). Serum concentrations of lutein and zeaxanthin were highest in France and Spain; beta-cryptoxanthin was highest in Spain and The Netherlands; trans-lycopene tended to be highest in Irish males and lowest in Spanish males; alpha-carotene and beta-carotene were higher in the French volunteers. Due to the study design, the concentrations of carotenoids and vitamins A, C and E represent physiological ranges achievable by dietary means and may be considered as 'reference values' in serum of healthy, non-smoking middle-aged subjects from five European countries. The results suggest that lutein (and zeaxanthin), beta-cryptoxanthin, total xanthophylls and gamma-tocopherol (and alpha- : gamma-tocopherol) may be important markers related to the healthy or protective effects of the Mediterranean-like diet.