Introduction: Infection with human papilloma virus (HPV) especially types 16 and 18 are associated with development of cervical intraepithelial neoplasia (CIN) and invasive carcinoma. HPV E6 and E7 which are consistently retained and expressed in the majority of CIN lesions and carcinomas are attractive targets for the immune system. Cytotoxic T lymphocytes (CTL) have been shown to clear viral infections and eliminate tumours. However there are few reports of human CTL specific for HPV E6 and E7. We have previously demonstrated HPV-specific CTL from one cancer patient following immunisation with recombinant vaccinia encoding HPV16/18 E6/E7 (TA-HPV). In this study we have investigated whether HPV-specific CTL responses occur naturally. We studied CTL responses against HPV16 and HPV18 E6/E7 in patients with CIN III and normal controls using the recombinant adenovirus (RAd) restimulation protocol. Here we report the detection and characterisation of CTL responses specific to both HPV16 and HPV18.Materials and Methods: Ten patients with CIN III and ten normal controls were studied. For CTL cultures responder PBMC were cocultured with irradiated autologous PHA blasts infected with recombinant adenoviruses (RAd) expressing HPV 16 E6/E7 or HPV 18 E6/E7. Cultures were restimulated after 1 week with RAd-infected autologous PHA blasts and supplemented with rhIL-2 and every 3-4 days thereafter. After another week cultures were restimulated as before and cultured for a 7 days. On day 21 effector cells were assayed against autologous and allogeneic B-LCL targets in a standard Cr release assay. Polyclonally expanded CTL lines were established from HPV-specific bulk cultures.Results: HPV specific CTL responses were detected in 610 patients. HPV 16 specific responses were detected in 3 patients and HPV 18 specific responses detected in 2 patients. In 1 patient both HPV 16 and HPV 18 specific responses were detected. No HPV 16 or HPV 18 CTL responses were detected in control subjects. Clonal CTL lines from one patient demonstrated HPV recognition restricted by at least 3 class 1 HLA alleles and retained their specificity in long term culture.Conclusion: This study demonstrates for the first time, naturally-occuring HPV-specific CTL induced by recombinant HPV E6/E7 proteins. These HPV specific CTL appear to be memory CTL as they were only detected in CIN III patients and not in normal controls. Specific CTL response to HPV is polyclonal and the recognition is restricted by multiple HLA alleles. These results have implications for immunotherapy of HPV-associated cervical cancer.