Gonococci, grown on agar, frequently give rise to opaque colonies. This opacity phenotype is associated with the presence of one or more outer membrane proteins of approximately 28,000 mol weight. These proteins are included within a class of proteins named proteins II. A method is described to isolate and purify the opacity-associated proteins from Neisseria gonorrhoeae. This method uses high concentrations of calcium and a zwitterionic detergent at pH 4.0. Under these conditions proteins II are readily solubilized from the outer membrane. Further purification is achieved by ion exchange and molecular sieve chromatography in the presence of the zwitterionic detergent. The opacity-associated proteins are very basic with isoelectric points varying between 9.0 to 10.0. Further evidence for their basic nature is their behavior on ion exchange chromatography and their amino acid composition.