The subunit structure of extracellular hemoglobin from the clam shrimp Caenestheria inopinata was studied using the method of cross-linking by bifunctional reagents followed by SDS/PAGE. Two phases were distinguished in the cross-linking by glutardialdehyde: a fast phase characterized by the appearance of two bands in electrophoresis corresponding to single polypeptide chains and cross-linked chain pairs, and a slow phase where five bands corresponding to even numbers, 2-10, of cross-linked polypeptide chains are observed. Theoretical curves for the distribution of protein among the various cross-linked species were calculated assuming allowed arrangements of ten identical subunits. Equally good descriptions of the cross-linking were provided by two models with dihedral symmetry: a ring arrangement with two types of alternating interactions and a two-layered eclipsed arrangement with one type of interaction between subunits from different layers and another between subunits within the same layer. A way out of the ambiguity was found by carrying out the fast phase of the cross-linking reaction with dimethyl-3,3'-dithiobispropionimidate.2HCl, a bifunctional reagent containing an S-S bond that can be cleaved by 2-mercaptoethanol, following up with glutardialdehyde in the slow phase. The observation in the presence of 2-mercaptoethanol of electrophoretic bands corresponding to trimeric and higher cross-linked polypeptide chain species rules out the alternating ring and confirms the two-layered eclipsed model. The arrangement of subunits found in this work from consecutive cross-linking can account satisfactorily for molecular profiles previously obtained from electron microscopy of Caenestheria hemoglobin.