Sedimentation equilibrium measurements and scanning transmission electron microscopy (STEM) mass mapping of the extracellular, hexagonal bilayer hemoglobin (HBL Hb) of the earthworm Lumbricus terrestris provided masses of 3.41 to 3.66 MDa and 3.56 (+/- 0.13) MDa, respectively. The Hb also contains 57.2 (+/- 6.0) moles of tightly bound Ca per mole of protein. The Hb and its subunits obtained by dissociation, in native, dehemed and reduced carbamidomethylated forms, were subjected to electrospray ionization mass spectroscopy (ESI-MS). Maximum entropy deconvolution identified three groups of peaks, at approximately 16 kDa, 24 to 32 kDa and approximately 53 kDa corresponding to the monomer subunit M (globin chain d), four linker subunits and the disulfide-bonded trimer T (globin chains a + b + c). Subunit M consisted of three components, d1 (15, 992.4), d2 (15, 978.0) and d3 (15, 962.1) (+/- 1.0 Da), with relative intensities 1.0:5:0.3, respectively. Subunit T consisted of four major components, T1 (52, 922.6), T2 (52, 760.0), T3 (52, 598.5) and T4 (52, 435.4) (+/- 4.0 Da), with relative intensities 0.6:1.0:0.2:0.7, respectively. ESI-MS of carbamidomethylated T, demonstrated that, unlike chains b (16, 254.4) and c (17, 289.2), chain a exists as a series of four, hexose-connected, glycosylated isoforms, a1 to a4 (19, 389.9, 19, 227.4, 19, 065.3 and 18, 902.9) (+/- 1.0 Da). The mass differences between the deglycosylated chain a (17, 524.0) and a1 to a4 correspond to glycan side-chains (GlcNAc)2 (Man)n (n = 6 to 9). Four groups of peaks were observed in the 24 to 32 kDa region. Linkers L1a (27, 540.8) and L1b (27, 702.4) (+/- 2.0 Da) are isoforms of L1 (25, 837.5 in N-deglycosylated Hb) with glycan side-chains (GlcNAc)2 (Man)n (n = 8,9). Linkers L2 (32, 104.3 (+/- 5.0) Da) and L3 (24, 912.9 (+/- 2.0) Da) occur as single species. Linkers L4a to L4c (24, 019.0, 24, 102.3 and 24, 169.9) (+/- 2.0 Da) with relative intensities 1.0:0.8:0.8, have not been identified previously. From ESI-MS relative intensities, L1:L2:L3:L4 = 0.6:0.4:1.0:0.5 and globin linker = 0.78:0.22. HPLC of Lumbricus Hb provided a globin linker = 0.73:0.27 (+/- 0.02) and a heme content of 2.52 (+/- 0.14) wt%. A model is proposed for the HBL structure, wherein 12 213.4 kDa dodecamers (144 globin chains, 2561 kDa) decorate a hexagonal framework of 36 linker chains (12L1 + 6L2 + 12L3 + 6L4) to provide a total mass of 3.531 MDa, each dodecamer being in contact with three linker subunits.