Primary squamous cell carcinoma of the endometrium (ESCC) is extremely rare. There has been no report of human papillomavirus (HPV) detected in ESCC. A 64-year-old Japanese female underwent total abdominal hysterectomy, bilateral salpingo-oophorectomy, and dissection of the pelvic, para-aortic, and bilateral inguinal lymph nodes. The tumor was confined to the endometrium, without invasion of myometrium or cervix. The endometrium was seen to be replaced by moderately differentiated squamous cell carcinoma, without any adenocarcinomatous element. Polymerase chain reaction amplification produced a 244- to 256-bp DNA fragment from the L1 region of the HPV genome. The amplification products were restricted, and the restricted fragment length polymorphism patterns were analyzed. The squamous cell carcinoma DNA was amplified for each exon, 5 through 8, of the p53 gene. The amplification products were used in single-stranded conformation polymorphism analysis. The tumor described showed the presence of HPV DNA. This amplified fragment was further purified by extraction from agarose gel, and the DNA was sequenced. Comparison of the fragment DNA sequence with known PV sequences showed that it had been amplified from HPV type 31. This is the first detection of HPV DNA in ESCC. Despite previous reports, HPV cannot not be excluded as a possible factor in the development of the malignancy.