Human papillomavirus type 18 (HPV18) is the second most common oncogenic HPV genotype, responsible for ϳ15% of cervical cancers worldwide. In this study, we constructed a full HPV18 transcription map using HPV18-infected raft tissues derived from primary human vaginal or foreskin keratinocytes. By using 5 rapid amplification of cDNA ends (RACE), we mapped two HPV18 transcription start sites (TSS) for early transcripts at nucleotide (nt) 55 and nt 102 and the HPV18 late TSS frequently at nt 811, 765, or 829 within the E7 open reading frame (ORF) of the virus genome. HPV18 polyadenylation cleavage sites for early and late transcripts were mapped to nt 4270 and mainly to nt 7299 or 7307, respectively, by using 3 RACE. Although all early transcripts were cleaved exclusively at a single cleavage site, HPV18 late transcripts displayed the heterogeneity of 3 ends, with multiple minor cleavage sites for late RNA polyadenylation. HPV18 splice sites/splice junctions for both early and late transcripts were identified by 5 RACE and primer walking techniques. Five 5 splice sites (donor sites) and six 3 splice sites (acceptor sites) that are highly conserved in other papillomaviruses were identified in the HPV18 genome. HPV18 L1 mRNA translates a L1 protein of 507 amino acids (aa), smaller than the 568 aa residues previously predicted. Collectively, a full HPV18 transcription map constructed from this report will lead us to further understand HPV18 gene expression and virus oncogenesis.Cervical cancer is a leading cause of death for women in the developing world, with about 493,000 new cases and nearly 273,000 deaths each year (www.who.int/hpvcenter/en/). Oncogenic human papillomavirus (HPV) infection is widely recognized as a principal cause of cervical, penile, and anal cancers (62). Among over 120 genotypes with human origin (3), infection with HPV16 and HPV18, the two most common oncogenic HPV types, leads to the development of ϳ70% of all cervical and other anogenital cancers (55, 62). HPV18 alone accounts for more than 15% of all cervical cancer cases worldwide (14). Persistent HPV16 infection is responsible for development of both cervical squamous cell carcinoma and adenocarcinoma. In contrast, persistent HPV18 infection is a preferential risk factor for the development of cervical adenocarcinoma (7, 10).HPVs are a group of small DNA viruses which have a high degree of conservation with respect to genome structure and organization as well as gene expression (44, 91). The circular, double-stranded viral genome is approximately 8 kb in size and contains eight open reading frames (ORFs) which are all transcribed from the same strand. In general, the viral genome can be divided into three major regions: an early region, a late region, and a long control region (LCR) or a noncoding region (NCR). The early region is positioned in the 5Ј half of the virus genome and encodes six common ORFs (E1, E2, E4, E5, E6, and E7) with regulatory functions in viral replication, gene expression, and pathogenesis. The late region lies dow...