The transgenic mouse models expressing beta s-globin genes do not fully exhibit the sickling phenotype, primarily as a result of the inhibition of beta s-chain-dependent polymerization by the mouse alpha-chains. The mouse alpha-chain differs from the human alpha-chain at 19 sequence locations. Of these, only alpha 78 and alpha 116 are the known hemoglobin (Hb) S polymer contact sites. To define whether the inhibition of polymerization by the mouse alpha-chain is solely a consequence of the differences at these two sites or additional sites of sequence differences are also involved, we have constructed chimeric alpha-chains by employing the alpha-globin semisynthetic reaction (Sahni, G., Cho, Y. J., Iyer, K. S., Khan, S. A., Seetharam, R., and Acharya, A. S. (1989) Biochemistry 28, 5456-5461). Mouse alpha 1-30 was spliced with human alpha 31-141 using endoproteinase Glu-C to generate a chimeric alpha-globin (alpha MH) containing eight of the 19 sequence differences of mouse alpha-globin. Similarly, human alpha 1-30 was spliced with mouse alpha 31-141 to generate another chimeric alpha-globin (alpha HM) containing 11 sequence differences. The respective chimeric globins were purified, reconstituted with heme and beta s-chain into tetrameric hemoglobin, and the tetramers were purified by ion-exchange chromatography. The inhibitory potential of the chimeric alpha MH-chain on the polymerization is 10-fold lower than that of the mouse alpha-chain. The absence of the alpha 31-141 region of the mouse alpha-chain relieves only a portion of the inhibition. The inhibitory potential of alpha MH contributed by the mouse alpha 1-30 segment is significant although none of the sequence differences in this segment are located at any of the implicated polymer contact sites. The chimeric alpha HM-chain also inhibits the polymerization, but the extent of inhibition is again lower (4-fold) than that of the full-length mouse alpha-chain. The results demonstrate that the inhibitory potential of mouse alpha-chains involves the sequence differences from both the alpha 1-30 and alpha 31-141 regions. Besides, since the sum of the inhibitory potential of either of these chimeric alpha-chains is lower than that of the intact mouse alpha-chains, we speculate that conformational changes that require the copresence of sequence differences in both portions of the mouse alpha-chain also contribute to the inhibitory propensity of the mouse alpha-chain.