Genital human papillomavirus (HPV) infection is generally considered to be sexually transmitted. However, nonsexual spread of the virus has also been suggested. The goal of this study was to assess: (a) the role of sexual intercourse in the transmission of HPV; (b) the determinants for seroconversion; and (c) the correlation between HPV DNA, abnormal cervical cytology, and serological response to HPV16. One hundred virgins and 105 monogamous women were randomly selected from a population-based cohort study in Copenhagen, Denmark, in which the women were examined twice with 2-year interval (interview, cervical swabs, Pap smear, blood samples). The presence of HPV DNA was determined by GP5+/6+ primers based HPV-PCR-EIA. HPV 16 virus-like particles (VLP) antibodies were detected by ELISA. All of the virgins were both HPV DNA negative and seronegative to VLP16, except for one woman who was weakly HPV 6 DNA positive. Only those virgins who initiated sexual activity became HPV DNA positive and/or VLP16 positive. The most important determinant of HPV DNA acquisition was the number of partners between the two examinations. The only significant risk factor for HPV 16 VLP seroconversion among women acquiring HPV DNA was HPV type. Our results show that sexual intercourse is important in the transmission of HPV, and that HPV 16 VLP seroconversion and the development of cervical lesions only occur after HPV transmission. Remarkably, no cervical lesions were found in HPV 16 DNA positive women who had seroconverted. Although based on small numbers, this may suggest that the development of antibodies had a protective effect.