The primary role of the water-soluble vitamin B2, i.e. riboflavin, in cell biology is connected with its conversion into FMN and FAD, the cofactors of a large number of dehydrogenases, reductases and oxidases involved in energetic metabolism, redox homeostasis and protein folding as well as in diverse regulatory events. Deficiency of riboflavin in men and experimental animal models has been linked to several diseases, including neuromuscular and neurological disorders and cancer. Riboflavin at pharmacological doses has been shown to play unexpected and incompletely understood regulatory roles. Besides a summary on riboflavin uptake and a survey on riboflavin-related diseases, the main focus of this review is on discovery and characterization of FAD synthase (EC 2.7.7.2) and other components of the cellular networks that ensure flavin cofactor homeostasis.Special attention is devoted to the problem of sub-cellular compartmentalization of cofactor synthesis in eukaryotes, made possible by the existence of different FAD synthase isoforms and specific molecular components involved in flavin trafficking across sub-cellular membranes.Another point addressed in this review is the mechanism of cofactor delivery to nascent apo-proteins, especially those localized into mitochondria, where they integrate FAD in a process that involves additional mitochondrial protein(s) still to be identified. Further efforts are necessary to elucidate the role of riboflavin/FAD network in human pathologies and to exploit the structural differences between human and microbial/fungal FAD synthase as the rational basis for developing novel antibiotic/antimycotic drugs.