The crucial roles of carotenoids and their metabolites in photooxidative protection and photosynthesis, not to mention nutrition, vision, and cellular differentiation, make them an important and complex class of biological pigments. Significant advances within the last few years have enhanced our understanding of the genetics and molecular biology of carotenoid biosynthesis in bacteria, fungi, algae, and plants. All of the genes involved in carotenoid biosynthesis from Rhodobacter capsulatus, an anoxygenic photosynthetic bacterium, and from several species of Erwinia, nonphotosynthetic bacteria, have been molecularly characterized. Recent studies have revealed that two early enzymes of carotenoid biosynthesis, geranylgeranyl pyrophosphate synthase and phytoene synthase, are structurally and functionally related in all carotenogenic organisms. In contrast, the subsequent conversion of phytoene, the first C(40) carotenoid, to beta-carotene requires two desaturases and one cyclase in oxygenic photosynthetic organisms (cyanobacteria, algae, and higher plants) but only one structurally distinct desaturase and a structurally distinct cyclase in other carotenogenic bacteria and in fungi. Studies of the enzymes that introduce oxygen-containing functional groups into carotenes to produce xanthophylls, the vast majority of all carotenoids, are still in their infancy. This review summarizes the most recent developments in carotenoid biosynthesis from a molecular genetic standpoint.