Over the past 60 years, Neisseria gonorrhoeae has acquired clinically significant resistance to sulfonamides, tetracyclines, penicillins, and ciprofloxacin. To determine U.S. trends in the prevalence of antimicrobial resistance of N. gonorrhoeae from 1988 to 2003. 16-year, multisite, sentinel surveillance for gonococcal isolate susceptibility through the Gonococcal Isolate Surveillance Project (GISP). Sexually transmitted disease clinics in 37 cities. Male patients with a total of 82,064 episodes of urethral gonorrhea. Primary outcome measures included percentage of gonococcal isolates resistant to antimicrobials used to treat gonorrhea, percentage of patients treated with specific antimicrobials for gonorrhea, and trends of these measures over time. The median age of patients was 26 years, and 74.1% of patients were African American. The proportion of men treated with penicillins for gonorrhea declined from 39.5% in 1988 to 0% in 1994, while the proportion of those receiving fluoroquinolone treatment increased from 0% in 1988 to 42.0% in 2003. Penicillin resistance peaked at 19.6% in 1991, then declined to 6.5% in 2003. Tetracycline resistance peaked at 25.8% in 1997 and declined to 14.4% in 2003. The first fluoroquinolone-resistant isolate was found in 1991. Nationally, 0.4% of isolates were fluoroquinolone-resistant in 1999 and were identified in 39% of GISP cities. By 2003, 4.1% of isolates were fluoroquinolone-resistant and were identified in 70% of GISP cities. Isolates with decreased susceptibility to ceftriaxone, cefixime, azithromycin, and spectinomycin remained rare. In 2001, 3 multidrug-resistant isolates with decreased susceptibility to cefixime were identified. Sentinel surveillance may not fully reflect trends for all patients with gonorrhea in the United States. Prevalence of penicillin resistance has declined in the years since gonorrhea treatment with penicillin was discontinued. Fluoroquinolone-resistant N. gonorrhoeae infections continue to increase at a time when fluoroquinolone use has increased. Ongoing nationwide and local antimicrobial susceptibility monitoring is crucial to ensure appropriate treatment of gonorrhea.