The PIK3CA gene encoding the p110α subunit of Class IA phosphatidylinositol 3-kinases (PI3Ks) is frequently mutated in human tumors. Mutations in the PIK3CB gene encoding p110β, the only other widely expressed Class IA PI3K, have not been reported. We compared the biochemical activity and transforming potential of mutant forms of p110α and p110β in a human mammary epithelial cell system. The two most common tumor-derived alleles of p110α, H1047R and E545K, potently activated PI3K signaling. Human mammary epithelial cells expressing these alleles grew efficiently in soft agar and as orthotopic tumors in nude mice. We also examined a third class of mutations in p110α, those in the p85-binding domain. A representative tumor-derived p85-binding-domain mutant R38H showed modestly reduced p85 binding and weakly activated PI3K/Akt signaling. In contrast, a deletion mutant lacking the entire p85-binding domain efficiently activated PI3K signaling. When we constructed in p110β a mutation homologous to the E545K allele of p110α, the resulting p110β mutant was only weakly activated and allowed minimal soft-agar growth. However, a gene fusion of p110β with the membrane anchor from c-Src was highly active and transforming in both soft-agar and orthotopic nude mouse assays. Thus, although introduction of activating mutations from p110α at the corresponding sites in p110β failed to render the enzyme oncogenic in human cells, the possibility remains that other mutations might activate the β isoform.