Carotenoids might lower the incidence of several diseases, yet the mechanisms governing their intestinal absorption are still poorly understood. The aim was to identify and study the main factors governing the transfer of carotenoids from emulsion lipid droplets to mixed micelles, presumed to be a key step in carotenoid absorption. An in vitro model was devised to measure the transfer, and a factorial design was applied to identify the main factors affecting the transfer. Experiments were then conducted to assess the effect of physiological variations of the main factors on the transfer efficiency. Finally, different carotenoids were simultaneously incorporated in emulsion lipid droplets to determine whether they interacted during the transfer. The factorial design gave three factors that significantly affected the transfer: type of carotenoid, pH, and bile lipid concentration. The transfer was (i) inversely related to carotenoid hydrophobicity, (ii) maximum between pH 6 and 7, (iii) maximum from 2 mmol/l bile salts, (iv) impaired by other carotenoids in the case of carotenes, but not in the case of xanthophylls. The transfer mainly depends on carotenoid hydrophobicity, pH, and bile lipid concentration. Physiological variations in pH and bile lipid concentration markedly affect the transfer. Both carotenes and xanthophylls can impair the transfer of carotenes, whereas they have apparently no effect on the transfer of xanthophylls.