Analytical dark-field scanning transmission electron microscopy (STEM) of freeze-dried unstained specimens of keyhole limpet hemocyanin (KLH ; from Megathura crenulata, a prosobranch gastropod) gave a molecular mass of 400 kDa for the subunit of KLHl and of 345 kDa for the subunit of KLH2, which confirms our published values from SDS/PAGE. Within the 400-kDa KLHl subunit we identified, by limited proteolysis, isolation of fragments and N-terminal sequencing, eight distinct 45 -60 kDa functional domains (termed l a through lh) and determined their sequential arrangement. The KLHl domains differ biochemically and immunologically from each other and from the previously characterized seven domains of KLH2 (termed 2a through 2g). Our partial amino acid sequences suggest that a domain, equivalent to the C-terminal domain lh, is missing in KLH2. This deficiency is believed to be genuine and not an artifact of the subunit preparation procedure, since STEM measurements of the native didecamers yielded a mass difference of about 800 kDa between KLHl and KLH2 (8.3 MDa versus 7.5 MDa), correlating with 20 copies of a functional l h domain. It was also shown that the KLHl didecamer can be rapidly split (minutes) into an almost homogeneous population of stable decamers by increasing the pH of the Triskaline stabilizing buffer (routinely pH 7.4), which contains 5 mM CaCl, and 5 mM MgCI,, to pH 8.5. Reformation of the didecamers occurred more slowly (days) upon dialysis against the pH 7.4 stabilizing buffer. Addition of 100 mM calcium and 100 mM magnesium ions to the pH 7.4 stabilizing buffer leads to the more rapid (overnight) formation of didecamers together with a significant number of previously unobserved KLHl multidecamers, which could be structurally distinguished from the established multidecamers of KLH2.