The brine shrimp, Artemia salina, produces at least three chemically and ontogenetically distinct extracellular hemoglobins (Hb-I, Hb-II and Hb-III). The estimated molecular weights of these hemoglobins are 240000-260000, containing 14 heme groups based on the iron and heme contents of a molar species. Hb-II, which corresponds to a minimal molecular weight of about 18 000 per heme [Moens, L. and Kondo, M. (1977) Biochem. J. 165, 111-119]. Denaturation of the reduced and alkylated hemoglobins with 8 M guanidine hydrochloride revealed apparently one polypeptide chain having a molecular weight of 126 000. Thus a single native hemoglobin molecule should be composed of two of these high-molecular-weight subunits each of which is bound with seven hemes. Upon sodium dodecyl sulfate/polyacryamide gel electrophoresis of either native hemoglobins or isolated subunits it was found that the 126 000-Mr polypeptide was cleaved specifically into two unequally-sized fragments of Mr 50 000 and 80 000. Further denaturation of native hemoglobins with urea at pH 2.5 or 11 followed by sodium dodecyl sulfate gel electrophoresis confirmed these results. The amino acid compositions determined for native Hb-II and its subunit and fragments are found to be very similar, implying that no specifically localized amino acid sequences are present and that the subunit globin chain could be composed of seven similar repeat units (Mr approximately 18 000) being linked covalently to one another. The amino acid compositions of Hb-I and Hb-III showed only minor differences to that of Hb-II.