Clathrin in coated vesicles is linked to transmembrane receptors by adaptor protein complexes. The Golgi-associated adaptor complex HA1 is a tetramer, made up of beta', gamma, 47-kDa, and 20-kDa subunits, whereas the tetrameric plasma membrane adaptor, HA2, contains alpha, beta, 50-kDa, and 16-kDa subunits (Ahle, S., Mann, A., Eichelsbacher, U., and Ungewickell, E. (1988) EMBO J. 7, 919-929). Here we report on the structural organization of adaptor subunits as revealed by proteolytic dissection. We show that the beta' and gamma subunits of HA1 are cleaved into 60-67-kDa "trunk" and 32-44-kDa "head" fragments. Interactions between adaptor subunits involve the trunk domains only. In overall organization of their domains, the Golgi and plasma membrane adaptors are very similar. The similarity encompasses also the location of phosphorylated serine residues in the alpha a, beta, beta', and gamma subunits, which are found in the head domains in all cases. In the alpha a and beta subunits they probably occur in the proline- and glycine-rich hinge region, which connects the head to the trunk. Identical adaptor fragments were obtained by controlled digestion of clathrin-coated vesicles. Under conditions that did not affect the integrity of the clathrin heavy chain, the adaptor head fragments were always quantitatively released from coated vesicles. The release of the bulk of the adaptors occurred concomitantly with the cleavage of their beta-type subunits (beta and beta') and under buffer conditions that prevent aggregation of adaptors. These observations taken together with the results of reconstitution experiments confirm and extend previous data (Ahle, S., and Ungewickell, E. (1989) J. Biol. Chem. 264, 20089-20093) which suggested that adaptors attach to clathrin through their beta-type (beta and beta') subunits. Moreover, high affinity interaction between adaptors and clathrin requires the participation of regions from both the head and trunk domains of the beta-type subunits.