Expression of a dominant negative atypical protein kinase C (aPKC), PKCζ, prevents nuclear translocation of extracellular regulated kinase 2 (ERK-2), p27 nuclear reduction, and DNA synthesis induced by estradiol in human mammary cancer-derived MCF-7 cells. aPKC action upstream of these events has been analyzed. In hormone-stimulated NIH 3T3 and Cos cells ectopically expressing human estrogen receptor alpha (hERα), aPKC is activated by phosphatidylinositol 3-kinase (PI 3-kinase) and, in turn, controls the Ras/MEK-1/ERK cascade. In MCF-7 and Cos cells stimulated by hormone, PI 3-kinase activates PKCζ by Thr410 phosphorylation. Serine phosphorylation of PKCζ is simultaneously induced. PKCζ activation leads to recruitment of Ras to a multimolecular complex that also includes hERα, Src, PI 3-kinase, and aPKC. We propose that PKCζ pushes Ras and the signaling complex close together in such a way that it facilitates the Src-dependent Ras activation. This activation is crucial for the interplay between estradiol-triggered signaling and cell cycle machinery.