Carotenoids are an excellent example of where poor understanding of food structure, complexity of behaviour during digestion, and inter-individual differences in response, lead to misinterpretation of study results. Four challenges associated with understanding and measuring carotenoid bioavailability are discussed: release of carotenoids from food structure and processing into an absorbable form (bioaccessibility), passage of carotenoids from gut lumen into the body (absorption), interpreting plasma response and inter-individual variation. Bioaccessibility of carotenoids is governed by characteristics of the food matrix, which affect the efficiency of physical, enzymic and chemical digestion. Carotenoids used as colorants are likely to be better absorbed because of the form in which they are dispersed in food. Extent of absorption of carotenoid supplements will depend on the proximity of dosing to the consumption of a fat-containing meal. Release of carotenoids from food plants occurs only when the plant cell is fractured and this occurs only during food preparation, processing and/or mastication, not during digestion. Following release from the food matrix, the major limiting factor is solubility of carotenoids in digesta. Absorption studies are best carried out by measuring chylomicron carotenoid excursion, with modelling of chylomicron turnover rate. In this way, inter-individual differences in lipoprotein metabolism can, in part, be taken into account before formulating conclusions on the rate and extent of absorption.