The effects of oral supplementation of a 30-mg dose of β-carotene on the plasma levels of carotenoids, tocopherols, and retinol were studied sequentially in 69 patients participating in a nine-month randomized placebo controlled trial conducted to examine efficacy of β-carotene to induce regression of cervical intraepithelial neoplasia. At each visit (baseline and 1.5, 3, 6, 9, 10.5, and 15 mo), blood samples were collected and the levels of six micronutrients were determined by high-performance liquid chromatography. No limitations or changes were introduced in each participant's dietary habits. Cervicovaginal lavage samples were also obtained at the same visit and assayed for the presence of human papillomavirus DNA by Southern blot hybridization and polymerase chain reaction. In the supplemented group, mean plasma β-carotene levels were significantly higher (p = 0.0001) than baseline and remained markedly elevated for 15 months. In the longitudinal analysis of the placebo group, there were no variations among individual mean plasma levels of β-carotene, α-carotene, lycopene, retinol, γ-tocopherol, or α-tocopherol, suggesting absence of seasonal or dietary changes. In the placebo group, cigarette smoking and steroid contraceptive use were significantly associated with low levels of plasma β-carotene (p = 0.05 and p = 0.012, respectively). However, in contrast, in the β-carotene-supplemented group, steroid contraceptive use had no influence on the plasma β-carotene levels. An additional noteworthy finding was that β-carotene supplementation did not reverse the depletion effect in smokers. There was no association between the plasma levels of these six micronutrients in women with cervical intraepithelial neoplasia and persistent human papillomavirus infection status in the placebo or the supplemented groups. Functional sequential nutrient interactions with each other or with other essential micronutrients and possible long-term toxicity need to be addressed in clinical trials.