PURPOSE: To investigate the outcome of HIV-seropositive patients under highly active antiretroviral treatment (HAART) with anal cancer treated with radiotherapy (RT) alone or in combination with standard chemotherapy (CT). PATIENTS AND METHODS: Clinical outcome of 81 HIV-seronegative patients (1988-2003) and 10 consecutive HIV-seropositive patients under HAART (1997-2003) that were treated with 3-D conformal RT of 59.4 Gy and standard 5-fluorouracil and mitomycin-C were retrospectively analysed. 10 TNM-stage and age matched HIV-seronegative patients (1992-2003) were compared with the 10 HIV-seropositive patients. Pattern of care, local disease control (LC), overall survival (OS), cancer-specific survival (CSS), and toxicity were assessed. RESULTS: RT with or without CT resulted in complete response in 100% of HIV-seropositive patients. LC was impaired compared to matched HIV-seronegative patients after a median follow-up of 44 months (p = 0.03). OS at 5 years was 70% in HIV-seropositive patients receiving HAART and 69% in the matched controls. Colostomy-free survival was 70% (HIV+) and 100% (matched HIV-) and 78% (all HIV-). No HIV-seropositive patient received an interstitial brachytherapy boost compared to 42% of all HIV-seronegative patients and adherence to chemotherapy seemed to be difficult in HIV-seropositive patients. Acute hematological toxicity reaching 50% was high in HIV-seropositive patients receiving MMC compared with 0% in matched HIV-seronegative patients (p = 0.05) or 12% in all HIV-seronegative patients. The rate of long-term side effects was low in HIV-seropositive patients. CONCLUSION: Despite high response rates to organ preserving treatment with RT with or without CT, local tumor failure seems to be high in HIV-positive patients receiving HAART. HIV-seropositive patients are subject to treatment bias, being less likely treated with interstitial brachytherapy boost probably due to HIV-infection, and they are at risk to receive less chemotherapy.