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To report long-term clinical outcome of topical 1% 5-fluoruracil (5-FU) as a sole treatment of ocular surface squamous neoplasia (OSSN).41 patients affected by OSSN were included. Each patient underwent full ophthalmological examination at baseline, with cytological or histological confirmation. Patients were treated by topical chemotherapy with 1% 5-FU four times a day for 4 weeks. One course was defined as 4 weeks of topical chemotherapy. Adjunctive courses were administered after 1 month of chemotherapy-free interval.Mean follow-up was 105±32 months (range 60–171 months). Complete tumour regression was achieved in 34 cases (83%) after a mean of 1.5 courses (range, 1–3 courses). Univariate analysis revealed that complete response was significantly related to tumour thickness <1.5 mm (p=0.005), lack of fornix or tarsal involvement (p=0.015 and p=0.009, respectively) and the absence of multifocality (p=0.002). Histopathological diagnosis (intraepithelial neoplasia vs squamous cell carcinoma, p=0.019) and American Joint Committee on Cancer (AJCC) classification (T1 vs T2 or T3) (p=0.028) were also related to incomplete tumour response. In a multivariate analysis, just tumour thickness >1.5 mm (p=0.045) and multifocality (p=0.023) were correlated with incomplete tumour response. Transient and reversible low-to-mild local side effects were documented in 19 (48%) eyes.Topical 5-FU, as a sole therapy, is a long-term safe and effective treatment for patients affected by preinvasive OSSN and for a limited proportion (50%) of invasive OSSN.