The protein folding field has undoubtedly benefited from studies on the folding mechanism of c-type cytochromes. Many different structural aspects of these small heme-containing proteins led this protein family to be considered a well established model system for such studies. In this chapter, we shall briefly describe some of the major results that have been obtained on the folding mechanism of this protein family and highlight unanswered questions. 1. Introduction Cytochromes c (cyt c) are small monomeric proteins of 80-120 residues involved in different and crucial aspects of cellular life, from electron transport processes to apoptosis. These heme-proteins show a typical α-helical fold that is recognized as a structural superfamily in protein classification tools such as SCOP (Andreeva et al. 2008) or CATH (Orengo et al. 1997). The three major α-helices (generally referred to as N-terminal helix, 60’-helix and C-terminal helix, following numbering of aminoacidic residues of horse heart cyt c) wrap around the heme group that is covalently linked to the protein via two thioether bonds between its vinyl groups and two cysteine residues in the conserved CysXaaXaaCysHis motif (Figure 1). Attachment of the heme group to the apoprotein in vivo is a complex post-translational process that involves different enzymatic activities (Ferguson et al. 2008). It is therefore not surprising that attempts to obtain properly folded recombinant holo cytochromes c by heterologous expression in E. coli, was unsuccessful for a long time; efficient production of recombinant prokaryotic holo cyt c in E. coli, is now generally accomplished under control of the E. coli enzymatic apparatus for heme attachment (Thöny-Meyer et al. 1995). The heme iron is always axially coordinated to the His residue of the CysXaaXaaCysHis motif on the proximal side. While the His ligand is maintained even under denaturing conditions, the distal ligand, generally Met, is inherently labile and readily displaced by other side-chains, such as a deprotonated His or Lys, which can become trapped during folding (Babul and Stellwagen 1971). The presence of a covalently bound heme can be considered as the privilege and disgrace of these small single-domain proteins: on one hand, it is an ideal natural quencher of the intrinsic fluorescence of the protein, it enables the breakage or formation of some individual bonds to be monitored (Gianni et al. 2003), and it represents a perfect tool for the design of photo-induced experiments to follow ultra-fast conformational transitions (Jones et al. 1993), (Hagen et al. 1996), (Yeh and Rousseau 1998), (Chang 2003). On the other hand, the presence of this large prosthetic group has often hindered a generalization of the rules emerging from in vitro folding studies on this system. In this chapter we report some of the key findings on the folding pathway of c-type cytochromes. We first describe the equilibrium studies on this protein family and then extend our discussion to kinetic measurements. Finally, we present the hypothesis of a common folding mechanism for all c-type cytochromes, and discuss the possibility to tune their folding pathways along different parallel routes by mutagenesis. Each section briefly outlines experimental methodologies and analytical approaches classically employed in protein folding studies.