We determined whether serum carotenoid or retinol concentrations were altered by beta-carotene supplementation in the Alpha-Tocopherol, Beta-Carotene Cancer Prevention Study and whether such effects were modified by alcohol consumption or cigarette use. Participants in this substudy were 491 randomly selected men aged 58-76 y from the metropolitan Helsinki study center [237 receiving supplemental beta-carotene (20 mg/d) and 254 not receiving such supplementation]. Dietary carotenoids, retinol, and alcohol, and serum beta-carotene, alpha-tocopherol, retinol, and cholesterol were assessed at baseline. After an average of 6.7 y of supplementation, serum was collected and carotenoid, retinol, and alpha-tocopherol concentrations were determined by HPLC. Serum carotenoid fractions were highly correlated with each other (P < or = 0.0001). Compared with the unsupplemented group, the beta-carotene group had significantly higher serum concentrations of beta-carotene (1483%), alpha-carotene (145%), and beta-cryptoxanthin (67%) (P < or = 0.0001). Retinol concentrations were 6% higher (P = 0.03) and lutein was 11% lower (P = 0.02) in the supplemented group. Serum lycopene, zeaxanthin, and alpha-tocopherol did not differ according to beta-carotene-supplementation status. Although these beta-carotene-group differences were not significantly altered by amount of alcohol consumption, higher consumption (> 12.9 g/d, median) was related to lower (10-38%) concentrations of carotenoids, particularly beta-carotene, alpha-carotene, and beta-cryptoxanthin, in both the supplemented and unsupplemented groups. Smoking status did not significantly influence the supplementation-related differences in serum carotenoid and retinol values but concentrations of carotenoids were generally highest in participants who quit smoking while in the study and lowest in current smokers of > or = 20 cigarettes/d. This study showed that serum concentrations of non-beta-carotene carotenoids are altered by long-term beta-carotene supplementation and confirms the adverse effects of alcohol and cigarette smoking on serum carotenoids.