Therapeutic inhibitors are being developed against the phosphoinositide 3-kinase (PI3K) pathway, the deregulation of which drives tumor growth and survival in many cancers. There are eight PI3Ks in mammals divided into three classes. Class IA PI3Ks (p110alpha, p110beta, and p110delta) are critical for cell growth and survival, with the p110alpha isoform implicated as the most important in carcinomas. In this study, we examined the effects of small-molecule inhibitors of class IA PI3Ks to explore the contributions of different isoforms in cancer cells. Similar responses were seen in cancer cells with wild-type or activated mutant PI3K genes treated with p110alpha/delta or p110alpha/beta/delta inhibitors in cell viability assays. In contrast, PTEN-negative cell lines tended to be less responsive (4-fold overall) to an inhibitor of p110alpha/delta versus p110alpha/beta/delta. Combining a p110alpha/delta inhibitor with a p110beta inhibitor resulted in comparable potency to the p110alpha/beta/delta inhibitor. The disparity in efficacy was confirmed in vivo. Pharmacodynamic biomarker analysis revealed that an inhibitor with insufficient potency against the p110beta isoform was less effective at inhibiting the PI3K pathway in PTEN-negative tumor xenografts. Our results imply that patients with PTEN-negative tumors may preferentially benefit from treatment with a class I PI3K inhibitor that is capable of inhibiting the p110beta isoform.