1. The globin chain components of Sprague-Dawley rat hemoglobin were obtained by reverse-phase HPLC which showed the presence of two alpha-chain and four beta-chains. 2. The accurate molecular weight of each globin chain was determined by means of electrospray mass spectrometry. Extensive mass spectrometric analysis on several enzymatic digests by fast atom bombardment mass spectrometry (FAB-overlapping) meant to determine the complete sequence of the alpha-major and of the four beta-globins. 3. The primary structure of the alpha-major globin was found in agreement with literature data (Garrick et al., 1975 Biochem. J. 149, 245-258; Chua et al., 1987). 4. Sequence analysis of the four beta-globin chains showed that amino acid differences are restricted to two protein portions: the region 22-25 and 123-125, the remaining portions of the molecule being unchanged in the four globins. Furthermore, all the amino acid replacements correspond to single point DNA mutations and (with the exception of the substitution Asp 22-->Asn in the beta 2-globin) involve uncharged substitutions.