The objective of this study was to assess the utility of a second generation human papillomavirus (HPV) virus-like particle (VLP)-based ELISA as an adjunct to HPV DNA testing to identify women at risk for high-grade cervical intraepithelial neoplasia (CIN). Participants provided blood, cervical samples and interviewer-obtained questionnaire information. HPV VLPs for types 16, 18, 33, 45 and 52 were produced using a baculovirus expression system. These highly purified VLPs were used in a polymer-based ELISA test. Cases with biopsy-confirmed CIN (CIN I, n = 237; CIN II, n = 56; CIN III, n = 48) and controls (n = 351) with normal Pap smears were tested for HPV DNA by PCR and serologic response to multiple oncogenic HPV VLPs. 258/341 (76%) of cases and 230/351 (65.5%) of control patients had any type of HPV VLP antibody (OR = 1.63, 95% CI 1.16-2.30). More cases were seropositive than controls for each individual HPV type (p < 0.001 for HPV types 16, 18, 33 and 45; p = 0.06 for HPV 52). Reactivity to an increasing number of different HPV type-specific VLPs are associated with high-grade CIN independent of HPV DNA status. HPV VLP assays may be useful as an adjunct to HPV DNA testing in a subset of patients that needs to be defined by further studies.