PTEN, a protein tyrosine phosphatase with homology to tensin, is a tumor-suppressor gene on chromosome 10q23. Somatic mutations in PTEN occur in multiple tumors, most markedly glioblastomas. Germ-line mutations in PTEN are responsible for Cowden disease (CD), a rare autosomal dominant multiple-hamartoma syndrome. PTEN was sequenced from constitutional DNA from 25 families. Germ-line PTEN mutations were detected in all of five families with both breast cancer and CD, in one family with juvenile polyposis syndrome, and in one of four families with breast and thyroid tumors. In this last case, signs of CD were subtle and were diagnosed only in the context of mutation analysis. PTEN mutations were not detected in 13 families at high risk of breast and/or ovarian cancer. No PTEN-coding-sequence polymorphisms were detected in 70 independent chromosomes. Seven PTEN germ-line mutations occurred, five nonsense and two missense mutations, in six of nine PTEN exons. The wild-type PTEN allele was lost from renal, uterine, breast, and thyroid tumors from a single patient. Loss of PTEN expression was an early event, reflected in loss of the wild-type allele in DNA from normal tissue adjacent to the breast and thyroid tumors. In RNA from normal tissues from three families, mutant transcripts appeared unstable. Germ-line PTEN mutations predispose to breast cancer in association with CD, although the signs of CD may be subtle.