Human serum albumin (HSA) participates in heme scavenging, the bound heme turning out to be a reactivity center and a powerful spectroscopic probe. Here, the reversible unfolding of heme-HSA has been investigated by H-1-NMR relaxometry, circular dichroism, and absorption spectroscopy. In the presence of 6 equiv of myristate ( thus fully saturating all available fatty acid binding sites in serum heme-albumin), 1.0 M guanidinium chloride induces some unfolding of heme-HSA, leading to the formation of a folding intermediate; this species is characterized by increased relaxivity and enhanced dichroism signal in the Soret region, suggesting a more compact heme pocket conformation. Heme binds to the folding intermediate with K-d = (1.2 +/- 0.1) x 10(-6) M. In the absence of myristate, the conformation of the folding intermediate state is destabilized and heme binding is weakened [K-d = (3.4 +/- 0.1) x 10(-5) M]. Further addition of guanidinium chloride ( up to 5 M) brings about the usual denaturation process. In conclusion, myristate protects HSA from unfolding, stabilizing a folding intermediate state in equilibrium with the native and the fully unfolded protein, envisaging a two-step unfolding pathway for heme-HSA in the presence of myristate.