Hexagonal bilayer hemoglobins (Hbs) are approximately 3.6-MDa complexes of approximately 17-kDa globin chains and 24-32-kDa, nonglobin linker chains in a approximately 2:1 mass ratio found in annelids and related species. Studies of the dissociation and reassembly of Lumbricus terrestris Hb have provided ample evidence for the presence of a approximately 200-kDa linker-free subassembly consisting of monomer (M) and disulfide-bonded trimer (T) subunits. Electrospray ionization mass spectrometry (ESI-MS) of the subassemblies obtained by gel filtration of partially dissociated L. terrestris and Arenicola marina Hbs showed the presence of noncovalent complexes of M and T subunits with masses in the 213. 3-215.4 and 204.6-205.6 kDa ranges, respectively. The observed mass of the L. terrestris subassembly decreased linearly with an increase in de-clustering voltage from approximately 215,400 Da at 60 V to approximately 213,300 Da at 200 V. In contrast, the mass of the A. marina complex decreased linearly from 60 to 120 V and reached an asymptote at approximately 204,600 Da (180-200 V). The decrease in mass was probably due to the progressive removal of complexed water and alkali metal cations. ESI-MS at an acidic pH showed both subassemblies to consist of only M and T subunits, and the experimental masses demonstrated them to have the composition M(3)T(3). Because there are three isoforms of M and four isoforms of T in Lumbricus and two isoforms of M and 5 isoforms of T in Arenicola, the masses of the M(3)T(3) subassemblies are not unique. A random assembly model was used to calculate the mass distributions of the subassemblies, using the known ESI-MS masses and relative intensities of the M and T subunit isforms. The expected mass of randomly assembled subassemblies was 213,436 Da for Lumbricus Hb and 204,342 Da for Arenicola Hb, in good agreement with the experimental values.