The class lb antigen HLA-G is expressed as a membrane-bound protein like classical class la molecules (WHLA-G) but, unlike typical class I, isalso expressed as a soluble protein (SaHLA-G) with a unique C terminus. Our results show that, similar to classical class I proteins, the membrane-bound form of HLA-G associated with TAP, as evidenced by the ability to immunoprecipitate HLA-G class I heavy chain with TAP antisera. In contrast, the soluble G protein did not appear to associate with TAP in the same manner, since similar immunoprecipltation experiments failed to detect soluble G complex. A detailed analysis of peptides bound to the soluble and membrane HLA-G proteins expressed in the B lymphoblastoid cell line 721.221 showed that, like class la complexes, both HLA-G proteins consist of heavy and light chains complexed with nonameric peptides in a 1: l: l ratio. The two proteins bind essentially the same set of peptides, which are derived from a variety of intracellular proteins and define a peptide motif for HLA-G. The peptides contain Leu at the C terminus and Pro or small hydrophobic amino acids in position 3 followed by Pro or Gly in position 4. The complexity of the bound peptides is lower than that found for some class la complexes, but is more similar to class la than to the limited repertoire of some murine class lb molecules.