H-2Kk is a transmembrane glycoprotein having the N-terminal region of the heavy chain exposed at the cell surface and the C-terminal region exposed at the cytoplasmic face in its native configuration in the plasma membrane. The configuration of H-2K in hposomes formed by detergent dialysis was investigated by using fluorescently labeled H-2 and Co2+ ions to quench fluorescence. H-2KK was incorporated into sealed lipid vesicles when deoxycholate was removed by dialysis from a mixture of protein and lipid. Including 20 mM carboxyfluorescein (CF) in the mixture prior to dialysis resulted in CF trapped inside the vesicles at concentrations where self-quenching occurred. Vesicles with CF trapped inside were shown to be osmotically active and impermeable to Na+ and Co2+ ions. In order to examine the configuration of H-2KK in these liposomes, the heavy chain was covalently labeled by using the sulfhydryl reactive fluorescent reagents fluores- fluorescein- 5-ylmaleimide (NFM) or 5-[[2-[(iodoacetyl)amino]-ethyl]amino]naphthalene-1-sulfonic acid (IAEDANS). In both cases, approximately equal amounts of fluorescent label were incorporated into the N- and C-terminal regions of the protein. Incorporation of the labeled H-2 into liposomes and examination of the effect of Co2+ on the fluorescence showed that all of the label was accessible to quenching by Co2+ and thus exposed on the outside of the liposome. The results demonstrate that the H-2KK is incorporated into these liposomes in a hairpin configuration, not in the transmembrane configuration found in native membranes. Implications of these results for the mode of incorporation of anchored membrane proteins into liposomes by detergent dialysis and for biological studies of H-2 antigen recognition by lymphocytes are discussed.