BACKGROUNDStudies regarding the performance of anal cytology in which both the screening test (cytology) and the diagnostic test (high‐resolution anoscopy [HRA]) are performed in all members of a screening population are rare. The authors evaluated the performance of liquid‐based anal cytology in a cohort of homosexual men in Sydney, New South Wales, Australia.METHODSThe Study of the Prevention of Anal Cancer (SPANC) is a 3‐year prospective study of the natural history of anal human papillomavirus infection in homosexual men aged ≥35 years. At baseline, all participants underwent a liquid‐based anal cytology test and HRA at the same clinical visit. Biopsies were obtained for histological assessment if lesions suspicious for human papillomavirus infection were visible during HRA. Using any cytological abnormality as the threshold, the sensitivity, specificity, and positive and negative predictive values were calculated against histologically diagnosed high‐grade squamous intraepithelial lesions (HSILs).RESULTSAmong 617 men recruited, the median age was 49 years (range, 35‐79 years) and 35.7% were positive for the human immunodeficiency virus. Overall, the sensitivity of cytology was 83.2%, the specificity was 52.6%, the positive predictive value was 45.8%, and the negative predictive value was 86.7%. Specificity improved with increasing age (P for trend =.041). Sensitivity was significantly higher in men with >1 anal octant of biopsy‐confirmed HSIL (92.9% vs 77.7%; P = .010), and in those who had ≥10 metaplastic cells present on their cytology slides (87.5% vs 70.2%; P = .007).CONCLUSIONSAnal cytology was found to have a higher specificity in older men while maintaining sensitivity. Sensitivity was higher among those with more extensive HSILs and men with metaplastic cells present on cytology. Cancer Cytopathol 2016;124:415–24. © 2016 American Cancer Society.