H4B and related tetrahydroproteins are cofactors for several enzymes and can be generators or scavangers of reactive oxygen species in cells. This review focuses on pterin radical formation in enzymes. The properties of pterin and pterin radicals that are generated in chemical systems are covered. Furthermore, function aspects of several H4B-utilizing enzymes are compared and contrasted to provide perspective for understanding H4B radical formation in enzymes.