The PI3K (phosphoinositide 3-kinase) pathway regulates cell proliferation, survival and migration and is consequently of great interest for targeted cancer therapy. Using a panel of small-molecule PI3K isoform-selective inhibitors in a diverse set of breast cancer cell lines, we have demonstrated that the biochemical and biological responses were highly variable and dependent on the genetic alterations present. p110alpha inhibitors were generally effective in inhibiting the phosphorylation of PKB (protein kinase B)/Akt and S6, two downstream components of PI3K signalling, in most cell lines examined. In contrast, p110beta-selective inhibitors only reduced PKB/Akt phosphorylation in PTEN (phosphatase and tensin homologue deleted on chromosome 10) mutant cell lines, and was associated with a lesser decrease in S6 phosphorylation. PI3K inhibitors reduced cell viability by causing cell-cycle arrest in the G(1) phase, with multi-targeted inhibitors causing the most potent effects. Cells expressing mutant Ras were resistant to the cell-cycle effects of PI3K inhibition, which could be reversed using inhibitors of Ras signalling pathways. Taken together, our data indicate that these compounds, alone or in suitable combinations, may be useful as breast cancer therapeutics, when used in appropriate genetic contexts.