Heme cofactors are found in a wide range of proteins, where they play various roles in e.g., steroid1 and bioactive lipid synthesis,2 energy transduction,3 gene regulation,4 cellular signaling,5 oxygen transport6 and antibiotic biosynthesis.7 The diversity of physiological functions performed by hemoproteins means that heme is among the most versatile of protein cofactors.8 Aside from electron transferase functions observed in respiratory cytochromes (e.g., mitochondrial cytochrome c ref. 9), several hemoprotein sensory or catalytic functions are recognized that involve the binding of gaseous ligands to the heme iron and/or the dissociation or switching of amino acid side chains as axial ligands to the iron.4,10 Moreover, heme-dependent activation of iron-bound dioxygen (as seen in e.g., nitric oxide synthase and the cytochromes P450) enables a broad repertoire of reactions, including hydroxylation, epoxidation, demethylation and carbon-carbon bond cleavage.11–13 This chapter reviews (i) the basic properties and synthesis of heme cofactors, (ii) the nature of their attachment to hemoproteins and the various types of protein scaffolds in which hemes are incorporated, (iii) exemplary functions of hemoproteins that demonstrate their broad range of biochemical functions, and (iv) analytical techniques that facilitate the understanding of the structural and redox characteristics of the protein-bound heme cofactor, and the mode of its ligation to the protein.