Some 700 carotenoids occur naturally in great structural diversity in the free form or as esters. Their biosynthesis de novo, however, occurs only in plants, algae, fungi and bacteria. Animals obtain their carotenoids from the food they consume. This chapter is not an exhaustive survey of carotenoid biosynthesis, but it integrates results from all kinds of investigations of reaction mechanisms, genes and enzymes to summarise the current state of knowledge. This is described in a series of stages (i.e. formation of the biological isoprene unit as isopentenyl diphosphate via mevalonate or by the mevalonate-independent pathway and the construction of the C40 carotenoid skeleton as phytoene, followed by desaturation [accompanied by isomerisation], cyclisation, hydroxylation and later modifications to generate the variety of carotenoid structures). The cleavage of carotenoids into smaller fragments that may have biological activity is also considered. Finally, the influence of environmental, nutritional and developmental factors on the accumulation of carotenoids, especially in ripening fruit and developing flowers, is outlined, and models of enzyme assemblies and their association with membranes are presented.