The primary objective of this study is to show the activation and analyze the regulation of the MEK- S6 kinase pathway in high-grade ovarian cancer. Phospho-ERK (pERK), a direct substrate of MEK and 2 phosphorylation sites on the ribosomal protein, S6, Ser235/236, and Ser240/244, which are both targeted by the MEK and PI3-kinase/AKT pathways, were analyzed in 13 cell lines, 28 primary cancers and 8 cases of cancer cells from ascites. In primary cancers, ERK and S6 phosphorylation was measured by immunohistochemistry (IHC). pERK, pS6, pAKT, and p4EBP1 were also measured by Western blotting (WB). The regulation of S6 phosphorylation by the MEK and PI3-kinase pathways was determined in ovarian cancer cell lines. We observed frequent pERK expression in primary ovarian cancers (100% by WB, 75% by IHC) but not in ovarian cancer cells from ascites (25% of cases by WB). The activation of the AKT pathway, measured by pAKT expression occurred in 7 cases of primary ovarian cancer by WB, but in none of the ascites samples. In ovarian cancer cell lines, the MEK pathway had a greater effect on S6 phosphorylation in cells without hyperactive AKT signaling. Our data suggest that MEK is a potential drug target in high-grade ovarian cancer, however, cancer cells with hyperactive AKT and cancer cells in ascites may be less responsive to MEK inhibition. The phosphorylation of S6 as a specific biomarker for either MEK or PI3-kinase pathway activation should be used with caution.