Ascites production is characteristic of late stage human epithelial ovarian cancer (EOC) and it correlates with tumor spread clinically. Ascites contributes to the microenvironment of EOC cells and promotes tumor development by mechanisms that are incompletely understood. Lysophosphatidic acid (LPA), a major tumor-promoting factor in EOC ascites, is an enzymatic product of autotaxin (ATX) and phospholipase A2 (PLA2) enzymes. The contribution of PLA2 activities to ovarian tumorigenesis was investigated. The quantitative measurement of PLA2 activities in ascites and tissues as well as assay conditions selective for PLA2 subtypes were optimized and validated. PLA2 activities correlated with tumor-promoting activates in cell-based and in vivo assays. High activities consistent with both cytosolic and calcium-independent PLA2 were found in human EOC ascites for the first time. Elevated PLA2 and ATX activities were also observed in EOC compared to benign tumors and normal tissues. Cell-free and vesicle-free (S4) human EOC ascites potently promoted proliferation, migration, and invasion of human EOC cells in a PLA2-dependent manner. LPA mediated a significant part of the cell-stimulating effects of ascites. S4 ascites stimulated tumorigenesis/metastasis in vivo, and methyl arachidonyl fluorophosphonate was highly effective in inhibiting EOC metastasis in mouse xenograft models. PLA2 activity was found in conditioned media from both EOC cells and macrophages. In addition, elevated PLA2 activities were detected in blood samples of several cancers, including ovarian, lung, and pancreatic cancers. Collectively, our work implies that PLA2 activity is a potential marker and therapeutic target in EOC, and possibly other cancers.