Mammalian myoglobins contain two tryptophanyl residues at the invariant positions 7 (A-5) and 14 (A-12) in the N-terminal region (A helix) of the protein molecule. The simultaneous substitution of both tryptophanyl residues causes an incorrect folding with subsequent loss of heme binding. The introduction of a indolic residue in different molecular regions, i.e. G, E, and C helix resulted in a not correctly folded protein, suggesting that the tryptophanyl residues are strong structural determinants.