A protein of about 20 kDa was extracted by sodium cholate (1%, w/v) from outer membranes of a strain of Neisseria gonorrhoeae, BS4 (agar), which is resistant to killing by human phagocytes. When the protein was purified by repeated fractionation on Sephadex G75, contamination with other outer-membrane proteins and lipopolysaccharide was negligible. The protein contained a full complement of amino acids, with high levels of glutamic acid. Carbohydrate, detected by the anthrone method and by sugar and hexosamine analysis, was present, but at very low levels. There was a significant content of fatty acids (about 5.7% of the protein), indicating a lipoprotein. The 20 kDa lipoprotein: (1) neutralized the ability of antiserum against whole organisms of BS4 (agar) to reduce the resistance of this strain to phagocyte killing; (2) evoked in mice an antiserum which reduced this resistance and immunoblotted only with 20 kDa lipoprotein in the cholate extract of outer membranes; and (3) promoted resistance to intracellular killing of an otherwise phagocyte susceptible gonococcal strain (BSSH). This is strong evidence that it is a determinant of gonococcal resistance to phagocyte killing.