The etiology and pathogenesis of esophageal squamous cell papillomas (ESCP), rare benign tumors of human esophagus, are still controversial. Chronic mucosal irritation and infection with human papillomaviruses (HPV) are two proposed etiologies. To investigate these hypotheses, we screened 29 ESCPs from 28 patients originating from Slovenia and Poland for HPV infection using in situ hybridization (ISH) and polymerase chain reaction (PCR). No evidence of HPV DNA was found using ISH. By PCR, the presence of HPV DNA was detected in only one lesion using two different HPV L1 consensus primer sets. The restriction fragment analyses of PCR product showed patterns unique to HPV type 6. All other ESCPs were successfully amplified only with internal control human beta-globin primers. Our results show that HPV DNA is not frequently detectable in ESCPs, even when highly sensitive methods like PCR are used and that other pathogenetic mechanisms are more important in the etiology of ESCPs.