We have evaluated a neutralizing antibody assay which uses human papillomavirus (HPV) type 16 (HPV-16) and HPV-18 pseudovirions carrying a secretory alkaline phosphatase reporter gene and which can potentially measure functionally relevant HPV type-specific neutralizing antibodies. The reproducibility of the assay was excellent; for HPV-16, the intra- and interassay kappa values were 0.95 and 0.90, respectively; and for HPV-18, the corresponding values were 0.90 and 0.90. This assay was used to describe the kinetics of the neutralizing antibody response in a cohort of 42 young women who were recruited soon after first intercourse and who first tested positive for HPV-16 DNA or HPV-18 DNA, or both, during follow-up. Most women seroconverted following the first detection of type-specific HPV DNA and remained seropositive until the end of follow-up. Our findings are broadly consistent with those of two other cohort studies which have measured the serological response following an incident infection by using the technically simpler virus-like-particle-based enzyme-linked immunosorbent assay.