Phosphatidylinositol 3-kinase (PI3K)/AKT pathway aberrations are common in cancer. By applying mass spectroscopy-based sequencing and reverse-phase protein arrays to 547 human breast cancers and 41 cell lines, we determined the subtype specificity and signaling effects of PIK3CA, AKT, and PTEN mutations and the effects of PIK3CA mutations on responsiveness to PI3K inhibition in vitro and on outcome after adjuvant tamoxifen. PIK3CA mutations were more common in hormone receptor-positive (34.5%) and HER2-positive (22.7%) than in basal-like tumors (8.3%). AKT1 (1.4%) and PTEN (2.3%) mutations were restricted to hormone receptor-positive cancers. Unlike AKT1 mutations that were absent from cell lines, PIK3CA (39%) and PTEN (20%) mutations were more common in cell lines than tumors, suggesting a selection for these but not AKT1 mutations during adaptation to culture. PIK3CA mutations did not have a significant effect on outcome after adjuvant tamoxifen therapy in 157 hormone receptor-positive breast cancer patients. PIK3CA mutations, in comparison with PTEN loss and AKT1 mutations, were associated with significantly less and inconsistent activation of AKT and of downstream PI3K/AKT signaling in tumors and cell lines. PTEN loss and PIK3CA mutation were frequently concordant, suggesting different contributions to pathophysiology. PTEN loss rendered cells significantly more sensitive to growth inhibition by the PI3K inhibitor LY294002 than did PIK3CA mutations. Thus, PI3K pathway aberrations likely play a distinct role in the pathogenesis of different breast cancer subtypes. The specific aberration present may have implications for the selection of PI3K-targeted therapies in hormone receptor-positive breast cancer.