To elucidate further the molecular pathogenesis of cervical cancer we sought to determine whether mutations in the PTEN tumor suppressor gene are a feature of these cancers. Genomic DNA was extracted from 67 primary cervical cancers and 9 immortalized cervical cancer cell lines. Using the polymerase chain reaction, the nine exons and intronic splice sites of the PTEN gene were amplified using 11 primer pairs. Single strand conformation polymorphism analysis was used to screen for mutations in the PTEN gene and variant bands were subjected to DNA sequencing. The primary cancers were also tested for the presence of HPV DNA. Mutations in the PTEN gene were not detected in any of the immortalized cell lines, but sequence alterations were noted in 4/67 (6%) primary cervical cancers. Three mutations resulted in frameshifts that predict truncated protein products, including two cases in which we found an identical 4-base-pair deletion in codons 318-319. In addition, a one-base-pair insertion in codon 320 was seen in another case. Finally, a missense mutation (G143 A) was observed immediately adjacent to the catalytic site of the phosphatase domain in exon 5. In two cases with PTEN mutations in which corresponding normal DNA was available, loss of the wild type allele was demonstrated. There was no apparent relationship between the presence of a PTEN mutation and pathologic features or the presence of human papillomavirus DNA. Although mutations in the PTEN gene are found in only a small fraction of cervical cancers, alterations of this tumor suppressor gene may play a role in the development of some of these cancers.