3-Phosphoinositide-dependent protein kinase 1 (PDK1) is a mediator of multiple signaling pathways coupled to growth factor receptor activation in human cancers. To evaluate the role of PDK1 in mammary gland oncogenesis, COMMA-1D mouse mammary epithelial cells were retrovirally transduced with PDK1, and transformation was measured by anchorage-independent growth in soft agar. PDK1-expressing cells exhibited a high degree of transformation that was associated with the activation of Akt1 and an elevation of protein kinase C (PKC ) expression. Cells overexpressing Akt1 did not exhibit anchorage-independent growth, whereas PKC overexpression produced significant transformation, although to a lesser extent compared with PDK1. Coexpression of Akt1 and PKC led to a more than additive effect on transformation activity. Isografts of either PDK1or PKC -expressing cells but not Akt1-expressing cells in syngeneic mice led to formation of poorly differentiated mammary carcinomas. PDK1 was highly expressed in a majority of human breast cancer cell lines. These results suggest that activation of PDK1 can lead to mammary tumorigenesis, in part through PKC , and that PDK1 expression may be an important target in human breast cancer.