A technique to detect Neisseria gonorrhoeae directly in clinical specimens was developed using a modified DNA-hybridization method. It uses the gonococcal cryptic plasmid as the radiolabeled probe, can detect as few as 100 colony-forming units of N gonorrhoeae or as little as 0.1 pg of purified gonococcal plasmid DNA, and is highly specific. This technique for differentiating between gonococcal and nongonococcal urethritis was evaluated in men with symptomatic urethritis in Seattle. Sixty-three (89%) of 71 who had cultures positive for N gonorrhoeae were also positive by DNA hybridization, and all 42 whose cultures were negative were also negative by DNA hybridization. Five of six isolates from patients who were positive by culture but negative by hybridization lacked the gonococcal cryptic plasmid and belonged to a unique auxo-type which requires proline, citrulline, and uracil for growth.