The involvement of organic radicals in biological catalysis was once a controversial topic. Today, however, examples of protein radicals, substrate radicals, and radical intermediates pervade biochemical mechanisms in a wide array of processes. Radical S-adenosyl-l-methionine (SAM) enzymes catalyze diverse and challenging reactions across all kingdoms of life. These reactions are initiated by transfer of an electron from a site-differentiated [4Fe-4S]+ cluster to a coordinated SAM, leading to homolytic cleavage of the S-C5′ bond of SAM and formation of the organometallic intermediate W, in which the SAM-derived adenosyl moiety is directly bound to the unique iron of the [4Fe-4S] cluster through an Fe-C5′ bond. Homolytic cleavage of the Fe-C bond liberates a 5′-deoxyadenosyl radical, which abstracts a hydrogen atom from substrate to initiate the substrate-based chemistry. We have used time-resolved freeze-quench trapping coupled to EPR and ENDOR spectroscopy to demonstrate the progression of radical SAM reactions through sequential radical intermediates, and provided important clues to how Nature uses inorganic chemistry to harness, with exquisite regioselective and stereoselective control, the highly reactive 5′-deoxyadenosyl primary carbon radical for initiation of radical-based chemical transformations essential to life. Studies of a wide range of radical SAM enzymes with functions ranging from microbial anaerobic metabolism, to biosynthesis of antibacterial and antiviral natural products, to roles in human disease, are providing new insights into the rich chemistry catalyzed by enzymes in this remarkable superfamily.