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High-resolution anoscopy is colposcopy of the anus after applying 3 percent acetic acid. High-resolution anoscopy with biopsy was used as the standard for detecting high-grade anal neoplasia and was compared to detection of high-grade anal neoplasia by anal cytology, human papillomavirus testing, or the combination.A total of 125 men who have sex with men (MSM) were enrolled from a group of MSM identified by random digit dialing: HIV-negative = 85, HIV-positive = 35, and unknown status = 5. A specimen was taken for anal cytology and human papillomavirus testing, followed by high-resolution anoscopy with biopsy of any lesions.Ninety-one percent of HIV-positive and 57 percent of HIV-negative MSM had anal human papillomavirus infection. In HIV-positive men the sensitivity of abnormal cytology to detect high-grade anal neoplasia was 87 percent, and in HIV-negative MSM it was 55 percent. Among HIV-negative men, 9 of 20 cases of high-grade anal neoplasia would have been missed because cytology was negative, but the addition of human papillomavirus positivity increased sensitivity for the combination to 90 percent.Sensitivity and specificity of anal cytology and human papillomavirus testing are different in HIV-positive and HIV-negative MSM for detecting high-grade anal neoplasia when patients have high-resolution anoscopy-guided biopsy of lesions. The optimum use of human papillomavirus testing has yet to be defined. High-resolution anoscopy is an effective tool for diagnosing high-grade anal neoplasia.