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 Calpha (PKCalpha) expression. Cells overexpressing Akt1 did not exhibit anchorage-independent growth, whereas PKCalpha overexpression produced significant transformation, although to a lesser extent compared with PDK1. Coexpression of Akt1 and PKCalpha led to a more than additive effect on transformation activity. Isografts of either PDK1- or PKCalpha-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 PKCalpha, and that PDK1 expression may be an important target in human breast cancer.