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, retinal, 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 less than or equal to 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 less than or equal to 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 ill current smokers of greater than or equal to 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.