Eight overlapping icosapeptides covering the entire sequence of the E4 protein of human papillomavirus type 16 (HPV-16), were prepared and tested for their reactivity with human sera in IgG-specific ELISA. The strongest reactivity of sera from HPV-16 DNApositive invasive cervical carcinoma (INCA) patients was detected with the peptide denoted 16/E4-6, covering amino acids 51 to 70. Subsequently nearly 200 sera were tested for the presence of the 16/E4-6-specific antibody. Reactivity was more frequent in cervical intraepithelial neoplasia patients and INCA patients than in matched control subjects. Sera from INCA patients were also tested for antibody reactive with peptide 16/E7-2 covering the major type-specific reactive region of the HPV-16 E7 protein. Only four of 13 sera possessing the 16/E4-6-specific antibody were reactive with the 16/E7-2 peptide.The development and evaluation of type-specific serological tests for diagnosis of human papillomavirus (HPV)-related cervical cancer has recently received much attention (Dillner et al., 1989;Dillner, 1990; JochmusKudielka et al., 1989;Jenison et al., 1989Jenison et al., , 1990Jenison et al., , 1991 Krchfi~k et al., 1990;Mann et al., 1990; Suchfinkovfi et al., 1990 Suchfinkovfi et al., , 1991 K6chel et al., 1991). Although the majority of the research has been focused on the E7 protein, the product of the E4 open reading frame (ORF), which is poorly conserved among different HPV types (Doorbar et al., 1989), has also emerged as a possible candidate antigen in such tests. The E4 protein of HPV-1 is expressed abundantly in HPV-l-associated warts (Croissant et al., 1985;Doorbar et al., 1986) and is believed to play a role in virus maturation. Although an abundant E4-specific mRNA spliced to the 5' end of the E10RF was found in genital warts (Chow et al., 1987) and in a human cell line immortalized by HPV-16 DNA (Diirst et al., 1987), little is known about the extent of E4 ORF expression in genital precancerous and cancerous lesions associated with HPV-16. Distribution of anti-E4 antibodies in human sera was first reported by JochmusKudielka et al. (1989) who used the HPV-16 E4 fusion protein as the antigen in Western blot (WB) assays. They have detected IgG anti-E4 antibody in 16% of invasive cervical carcinoma (INCA) patients and in 8% of matched control women; however, up to 40% anti-E4-positive sera were found among healthy teenagers and among women suffering from cervical intraepithelial neoplasia (CIN). On the basis of these data the authors 0001-0507 © 1992 SGM concluded that the presence of E4-specific antibody correlates more closely with virus replication than with the development of cancer. K6chel et al. (1991) who also employed HPV-16 fusion proteins as antigens in WB detected anti-E4 antibody in three out of four genital carcinoma patients but not in any of the 63 healthy women tested. Using an ELISA with synthetic peptides derived from the ORF of HPV-16 E4, Dillner (1990) found the major IgG-and IgA-reactive epitope(s) in a peptide covering ami...