A wide variety of tumours show PIK3CA mutations leading to increased phosphatidylinositol-3 kinase (PI3K) activity. We have determined the frequency of PIK3CA mutations in exons 9 and 20 that has previously been reported as mutational hotspot regions in distinct tumour models. One hundred and fifty gastrointestinal carcinomas (47 gastric and 103 colorectal) that were characterised for MSI status (76 MSI and 74 MSS) by PCR-SSCP sequencing were evaluated. We also analysed the association between PIK3CA mutations and KRAS or BRAF mutations. PIK3CA mutations in exons 9 and 20 were present in 13.6% and 10.6% of colorectal and gastric carcinomas, respectively. No differences in frequency and type of PIK3CA mutations were found between MSI and MSS colorectal carcinomas. All gastric carcinomas with PIK3CA mutations were MSI. The number of cases harbouring concomitant PIK3CA and KRAS or BRAF mutations was higher in colorectal than in gastric carcinomas (P = 0.016). In colorectal carcinoma, PIK3CA mutations occur preferentially together with activating KRAS-BRAF mutations (MSI and MSS) while in gastric carcinomas PIK3CA mutations tend to occur as isolated events (MSI).