Germline mutations in PTEN can predispose people to Cowden syndrome (CS) and Bannayan-Ruvalcaba-Riley (BRR) syndrome, rare, autosomal dominantly inherited neoplastic disorders. To determine whether germline mutations in PTEN contribute to genetic predisposition to multiple primary tumours within the general population, we conducted a nested case-control study, among 32 826 members of the prospective Nurses' Health Study cohort; cases were women with more than one primary tumour at different anatomical sites. We screened all nine exons of PTEN and flanking intronic splice sites for all 103 eligible cases using SSCP and sequencing. We observed two novel germline heterozygous missense mutations in exon 5 in five of the cases; three were V119L and two were V158L. Neither mutation was observed in 115 controls free of diagnosed cancer (p = 0.02). Both mutants showed partial tumour suppressor activity when compared to wild type PTEN when transfected into a PTEN null breast cancer cell line. The phenotype was cell line specific suggesting that genetic background affects growth suppression activity of the mutants. These data provide evidence that germline mutations in PTEN may be a more frequent predisposing factor for cancers in women than previously suggested.