BACKGROUND Nasopharyngeal carcinoma (NPC) is a common cancer in Southeast Asia and is frequently associated with Epstein-Barr virus (EBV). Human papilloma virus (HPV) is an epitheliotrophic oncogenic virus that has been detected in a variety of head and neck tumors including NPC. This retrospective study was undertaken to investigate the prevalence of EBV and HPV infection subtypes 6/11 and 16/18 in 20 patients with NPC. METHODS In situ hybridization for EBV-encoded RNA (EBER) and tyramid signal amplification of ISH for HPV DNA subtypes 6/11 and 16/18 was performed to evaluate the prevalence of EBV and HPV latency infection among Iranian Patients with NPC. RESULTS 16 cases were classified as WHO type III (undifferentiated carcinoma) and 4 as WHO type II (non-keratinizing SCC). EBER-ISH was positive in 19 (95%) of NPCs evaluated and in one metastases from cervical primary, included in this series. Two of 20 NPC (10%) contained HPV 6/11 sequences and two of 20 NPC (10%) contained HPV 16/18 sequences, and combined EBV and HPV infection was detected in 3 of the 20 (15%) patients. CONCLUSIONS Our data indicated that EBV is closely associated with NPC in Iran. In addition, a low percentage of EBV positive NPC contained HPV sequences. The significance of coexistence of EBV and HPV in NPC requires further study.