We examined 341 paraffin-embedded cervical tissues for human papillomavirus (HPV) DNA by in situ hybridization. The genital lesions examined represented tissue biopsies from two temporally distinct populations (1964 to 1965 and 1988 to 1989). Biotinylated probes to 14 different HPV types were used in our analysis: HPV types 6, 11, 16, 18, 31, 33, 35, 42, 43, 44, 45, 51, 52, and 56. The number of HPV DNA-positive specimens and the distributions of HPV types were similar for these two populations. Human papillomavirus DNA sequences were detected in approximately 50% of the tissues from each time period. Of the low-grade lesions (condyloma/cervical intraepithelial neoplasia 1 [CIN 1]) 52% (1964 to 1965) and 35% (1988 to 1989) were positive for HPV DNA by in situ hybridization. Among the high-grade lesions (CIN 2/CIN 3), 41% (1964 to 1965) and 67% (1988 to 1989) had detectable HPV sequences. Approximately 15% of the tissues with minimal histopathologic changes also contained HPV DNA. Human papillomavirus types 16 and/or 18 were the most common viral types in lesions from both time periods, followed by types 31/33/35; 6/11, 51/52; and 42/43/44, 45/46. Types 16 and/or 18 were strongly associated with high-grade lesions. Five percent of the HPV-positive lesions demonstrated evidence of multiple infections. Our results indicate that HPV DNA sequences can be detected readily by in situ hybridization in archival materials, even those prepared more than 25 years ago. In addition, analysis of HPV type distributions demonstrates that recently isolated HPV types (42, 43, 44, 45, 51, 52, and 56) were equally represented in tissues from both time periods.