Persistent human papillomavirus (HPV) infection of the uterine cervix is a risk factor for progression to high-grade squamous intraepithelial lesions. Detection in consecutive genital samples of HPV-16 DNA, a frequently encountered HPV type, may represent persistent infection or reinfection. We undertook a study using PCR–single-strand conformation polymorphism (SSCP) analysis and sequencing of PCR products (PCR-sequencing) to determine if consecutive HPV-16-positive samples contained the same HPV-16 variant. Fifty women (36 human immunodeficiency virus [HIV] seropositive, 14 HIV seronegative) had at least two consecutive genital specimens obtained at 6-month intervals that contained HPV-16 DNA as determined by a consensus L1 PCR assay. A total of 144 samples were amplified with two primer pairs for SSCP analysis of the entire long control region. Fifteen different SSCP patterns were identified in our population, while 22 variants were identified by PCR-sequencing. The most frequent SSCP pattern was found in 75 (53%) samples from 27 (54%) women. The SSCP patterns obtained from consecutive specimens were identical for 46 (92%) of 50 women, suggesting persistent infection. Four women exhibited in consecutive specimens different HPV-16 SSCP patterns that were all confirmed by PCR-sequencing. The additional information on the nature of persistent infection provided by molecular variant analysis was useful for 6% of women, since three of the four women who did not have identical consecutive specimens would have been misclassified as having persistent HPV-16 infection on the basis of HPV typing.