Background: Asymmetric alpha-carotene, a provitamin A carotenoid, is cleaved to produce retinol (vitamin A) and alpha-retinol (with negligible vitamin A activity). The vitamin A activity of alpha-carotene-containing foods is likely overestimated because traditional analytic methods do not separate alpha-retinol derivatives from active retinol. Objective: This study aimed to accurately characterize intestinal alpha-carotene cleavage and its relative contribution to postprandial vitamin A in humans after consumption of raw carrots. Design: Healthy adults (n = 12) consumed a meal containing 300 g raw carrot (providing 27.3 mg beta-carotene and 18.7 mg alpha-carotene). Triglyceride-rich lipoprotein fractions of plasma were isolated and extracted, and alpha-retinyl palmitate (alpha RP) and retinyl palmitate were measured over 12 h postprandially via high-performance liquid chromatography-tandem mass spectrometry. The complete profile of all alpha-retinyl esters and retinyl esters was measured at 6 h, and total absorption of alpha- and beta-carotene was calculated. Results: alpha RP was identified and quantified in every subject. No difference in preference for absorption of beta- over alpha-carotene was observed (adjusting for dose, 28% higher, P = 0.103). After absorption, beta-carotene trended toward preferential cleavage compared with alpha-carotene (22% higher, P = 0.084). A large range of provitamin A carotenoid conversion efficiencies was observed, with alpha-carotene contributing 12-35% of newly converted vitamin A (predicted contribution = 25.5%). In all subjects, a majority of alpha-retinol was esterified to palmitic acid (as compared with other fatty acids). Conclusions: alpha-Retinol is esterified in the enterocyte and transported in the blood analogous to retinol. The percentage of absorption of alpha-carotene from raw carrots was not significantly different from beta-carotene when adjusting for dose, although a trend toward higher cleavage of beta-carotene was observed. The results demonstrate large interindividual variability in alpha-carotene conversion. The contribution of newly absorbed alpha-carotene to postprandial vitamin A should not be estimated but should be measured directly to accurately assess the vitamin A capacity of alpha-carotene-containing foods.