The PIK3CA gene, encoding the p110α catalytic subunit of Class IA PI3Ks (phosphoinositide 3-kinases), is frequently mutated in many human tumours. The three most common tumour-derived alleles of p110α, H1047R, E542K and E545K, were shown to potently activate PI3K signalling in human epithelial cells. In the present study, we examine the biochemical activity of the recombinantly purified PI3K oncogenic mutants. The kinetic characterizations of the wt (wild-type) and the three ‘hot spot’ PI3K mutants show that the mutants all have approx. 2-fold increase in lipid kinase activities. Interestingly, the phosphorylated IRS-1 (insulin receptor substrate-1) protein shows activation of the lipid kinase activity for the wt and H1047R but not E542K and E545K PI3Kα, suggesting that these mutations represent different mechanisms of lipid kinase activation and hence transforming activity in cancer cells.