Anal cytology has been suggested as a screening test for the anal cancer precursor high-grade squamous intraepithelial lesion (HSIL). We aimed to assess the prevalence and predictors of initial unsatisfactory anal cytology tests (‘unsats’). The Study of the Prevention of Anal Cancer is a natural history study of anal human papillomavirus (HPV) and precancerous lesions among gay and bisexual men (GBM) of at least 35 years in Sydney, Australia. At each study visit, an anal swab is collected for cytological testing. Unsats are defined as slides with fewer than 2000 nucleated squamous cells and no abnormal cells. Among 617 GBM enrolled, the median age was 49 (range: 35–79) years and 220 (35.7%) were HIV positive. Initial unsats occurred in 61 (9.9%, 95% confidence interval: 7.6–12.5%), and 29 (4.7%, 95% confidence interval: 3.2–6.7%) remained unsatisfactory on repeat cytology. Initial unsats were associated with fewer lifetime anal-receptive partners with a condom (P=0.007); fewer recent anal-receptive sexual partners without a condom (P=0.005); never having had anal chlamydia (P=0.023) or gonorrhea (P=0.003); HIV-negative status (P=0.002); fewer total (P=0.002), low-risk (P=0.005), and high-risk (P=0.015) HPV types detected; lack of anal HPV18 detection (P=0.001); never having anally douched (P<0.001); and douching with soapy water (P=0.009) among those who douched. Unsats were less common among those with histologic HSIL (P=0.008) and nonsignificantly less common among those with fewer anal canal octants affected by HSIL (P=0.080), but were more common among those who felt more nervous (P=0.020) during the examination. Our findings suggest that unsats are more common among GBM with less receptive anal sexual experience. Avoiding douching with soapy water and strategies to aid patient relaxation during sampling may reduce the unsat rate.