G onococcal pili, pure by the criteria of electron microscopic examination and polyacrylamide gel electrophoresis in sodium dodecyl sulfate, have been prepared by repeated cycles of precipitation with 0.1 M MgCl₂, followed by dissolution in 0.01 M Tris pH 8, 0.01 M NaN₃. Using a fluorescein-conjugated antibody prepared to pili from a single strain, pilar antigen(s) was found to be present in each of 18 strains of gonococci tested, and absent from strains of pilated meningococci, nonpathogenic Neisseria sp., and Escherichia coli. Purified pili, labeled with ¹²⁵I were used in an antigen binding assay to quantitatively measure antibody to pili in rabbit sera and in 561 human sera. The range of antibody activity for 133 persons unlikely to have experienced gonorrhea was 0.1-1.6 μg/ml with a geometric mean of 0.5 μg/ml. This geometric mean antibody activity was significantly lower than the geometric mean for asymptomatically infected males (1.0 μg/ml, P < 0.002), males with symptomatic gonococcal anterior urethritis (1.6 μg/ml, P < 0.001), or asymptomatically infected females (4.2 μg/ml, P < 0.001). Antibody appeared in elevated levels (> 1.6 μg/ml) 2-3 wk after infection and returned toward control levels 1 or more months after treatment. Antibody levels higher than 1.6 μg/ml were found in 26 (50%) of 52 males with gonococcal anterior urethritis, in 10 (33%) of 30 males with asymptomatic urethral infection and in 50 (89%) of 56 asymptomatically infected females. In a high-risk group of 103 females for whom culture results and antibody to pili were compared, 58 (57%) had elevated antibody levels to pili and 86% of the infected females were within this seropositive group. The antigen binding assay may provide a means to detect asymptomatic gonococcal infection in women.