Attachment of Neisseria gonorrhoeae to HeLa cells was assessed by a technique using double radioisotopic labeling. Piliated, virulent bacteria from colony type 2 attached to HeLa cells to a greater extent than nonpiliated, avirulent bacteria from colony type 4. Maximal attachment rates for bacteria from both colony types occurred during the early incubation periods at 37 degrees C, and the HeLa cells appeared saturated at 4 h. Attachment was maximum at pH 6.5 and dependent upon the multiplicity of infection. Treatment of the HeLa cells with trypsin diminished the degree of attachment, but this effect substantially disappeared by 24 h after trypsin treatment. Scanning electron microscopy revealed bacteria of colony types 2 and 4 adhered to the HeLa cell surface. Thin-section transmission electron microscopy showed that bacteria were associated with the surface of the HeLa cell but not ingested.