The α- and β-subunits of human hemoglobin consist of the modules M1, M2 + M3, and M4, which correspond to the exons 1, 2, and 3, respectively (Go, M. (1961) Nature 291, 90-92). To gain further insight into functional and structural significance of the modules, we designed two kinds of chimeric hemoglobin subunits (chimeric ααβ-and ββα-subunits), in which the module M4 was replaced by the partner subunits. CD spectra in the far-UV region showed that the secondary structure of the chimeric ααβ-subunit drastically collapsed, while the chimeric ββα-subunit conserved the native globin structure (Wakasugi, K., Ishimori, K., Imai, K., Wada, Y., and Morishima, I. (1994) J. Biol. Chem. 269, 18750-18756). SAXS data also suggested a partially disordered structure of the chimeric ααβ-subunit. Based on tryptophan fluorescence spectra and computer modeling from x-ray structures of native globins, steric constraint between Trp14 and Tyr125 would be induced in the chimeric ααβ-subunit, which would perturb the packing of the A- and H-helices and destabilize the globule structure. On the other hand, such a steric constraint was not found for the counterpart chimeric subunit, the ββα-subunit. The different stabilities of these module-substituted globins imply that modules would not always be stable 'structural' units, and interactions between modules are crucial to construct stable globin subunits.