Oncogenic human papillomavirus (HPV) infection is a necessary cause of cervical cancer. Therefore, vaccination to prevent or eliminate HPV infection could reduce the incidence of cervical cancer. A fusion protein comprising HPV16 L2, E6, and E7 is a candidate combination preventive and therapeutic HPV vaccine. The L1- and L2-specific and neutralizing serum antibody titers and peripheral blood mononucleocyte antigen-specific proliferative responses generated by vaccination thrice at monthly intervals with HPV16 L2E7E6 were compared in two studies: a phase I randomized double-blind placebo controlled dose escalation trial in 40 healthy volunteers and a phase II trial of HPV16 L2E7E6 at the maximum dose in 29 women with high-grade anogenital intraepithelial neoplasia (AGIN). Vaccination of healthy volunteers induced L2-specific serum antibodies that were detected 1 month after the final vaccination (P(binomial) < 0.001). There was a significant trend to seroconversion for HPV16 and HPV18 neutralizing antibodies with increasing vaccine dose (P = 0.006 and P = 0.03, respectively). Seroconversion for HPV18 neutralizing antibodies showed a significant positive trend with increasing dose (P = 0.03) and was associated with seroconversion for HPV16 neutralizing antibodies (P(exact) = 0.04). The antigen-specific proliferative response of vaccinated healthy volunteers also showed a significant trend with increasing vaccine dose (P = 0.04). However, AGIN patients responded less effectively to vaccination than healthy patients for induction of HPV16 L2-specific antibody (P < 0.001) and proliferative responses (P < 0.001). Vaccination of healthy volunteers thrice with 533-mug HPV16 L2E7E6 at monthly intervals induced L2-specific serum antibodies that neutralized across papillomavirus species. Responses in AGIN patients were infrequent.