In the genes of alpha- and beta-subunits of hemoglobin, Go showed that modules F1, F2 + F3, and F4 correspond to exons 1, 2, and 3, respectively (Go, M. (1981) Nature 291, 90). The analysis of the correlation of function with its exon pattern showed that the residues associated with the defined function are concentrated in the specific exons encoding the "module" (Eaton, W.A. (1980) Nature 284, 183). To investigate the functional and structural significance of the "modular structure," we engineered a "chimera" subunit, in which module F4 of the beta-subunit was replaced by that of the alpha-subunit by use of mutagenesis. The NMR and resonance Raman spectra of the isolated "chimera beta alpha-subunit" have revealed that it has a beta-subunit-like heme environmental structure. However, the gel chromatography and NMR spectra of mixtures of the chimera and native subunits clearly showed that the chimera beta alpha-subunit binds specifically to the beta-subunit to form a heterotetramer, not to the alpha-subunit. These results led us to conclude that the predominant role of the module F4 is the subunit association and suggest that the modules are structural and functional units that have advantages in producing stable functional proteins.