The incidence of invasive anal squamous cell carcinoma, a human papilloma virus (HPV) related cancer, is on the rise, especially in HIV positive men who have sex with men (MSM). Like cervical cancer, anal cancer is associated with precursor lesions detectable on exfoliative cytology as squamous intraepithelial lesions and on biopsy as intraepithelial neoplasia. Anal–rectal cytology screening programs, similar to cervical cytology screening programs, have been developed in an effort to detect and to eradicate precursor lesions prior to progression to invasive squamous cell carcinoma. Either conventional or liquid-based anal–rectal cytology specimens are acceptable, but liquid-based specimens are preferred. Specimens may be collected by health care professionals or by patients. A minimum of 2,000–3,000 nucleate squamous cells should comprise adequate specimens. Diagnostic terminology as defined by the Bethesda System for Reporting Cervical Cytology (TBS 2001) should be used. Sensitivity and specificity of a single anal–rectal cytology specimen is comparable with that of a single cervical cytology test, but cytological interpretations do not always correlate with lesion severity. Patients with atypical squamous cells of undetermined significance (ASC-US) or worse should be referred for anoscopy. Diagn. Cytopathol. 2010. © 2009 Wiley-Liss, Inc.