Riboflavin is biosynthesized from GTP and ribulose 5-phosphate. Whereas the early reactions conducing to 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione 5'-phosphate show significant taxonomic variation, the subsequent reaction steps are universal in all taxonomic kingdoms. With the exception of a hitherto elusive phosphatase, all enzymes of the pathway have been characterized in some detail at the structural and mechanistic level. Some of the pathway enzymes (GTP cycloyhdrolase II, 3,4-dihydroxy-2-butanone 4-phosphate synthase, riboflavin synthase) have exceptionally complex reaction mechanisms. The commercial production of the vitamin is now entirely based on highly productive fermentation processes. Due to their absence in animals, the pathway enzymes are potential targets for the development of novel anti-infective drugs.