Carotenoids are important photosynthetic pigments that play key roles in light harvesting and energy transfer, photoprotection, and in the folding, assembly, and stabilization of light-harvesting pigment-protein complexes. The genetically tractable purple phototrophic bacteria have been useful for investigating the biosynthesis and function of photosynthetic pigments and cofactors, including carotenoids. Here, we give an overview of the roles of carotenoids in photosynthesis and of their biosynthesis, focusing on the extensively studied purple bacterium Rhodobacter sphaeroides as a model organism. We provide detailed procedures for manipulating carotenoid biosynthesis, and for the preparation and analysis of the light-harvesting and photosynthetic reaction center complexes that bind them. Using appropriate examples from the literature, we discuss how such approaches have enhanced our understanding of the biosynthesis of carotenoids and the photosynthesis-related functions of these fascinating molecules.