High levels of tumour kallikrein-related-peptidase 4 (KLK4) and KLK7 are associated with a poor outcome for women with serous epithelial ovarian cancer (EOC), for which peritoneal dissemination and chemoresistance are key events (1). As accumulation of fluid in the peritoneal cavity, ascites, is a common feature of EOC, we wished to determine the role of KLKs in EOC dissemination and chemoresistance in this microenvironment. We examined KLK4- and KLK7-transfected SKOV3 EOC cells in 3-dimensional (3D) suspension culture to mimic the ascites fluid. KLK4-SKOV3 cells formed multicellular aggregates (MCAs) as seen in ascites, as did SKOV3 cells treated with active KLK4. MCA formation was reduced by treatment with a KLK4 blocking antibody, or the selective sunflower trypsin KLK4 inhibitor (SFTI-FCQR). SFTI-FCQR also reversed paclitaxel resistance of KLK4-MCAs. KLK4-MCAs formed larger cancer cell foci in mesothelial cell monolayers than those formed by vector and native SKOV3 cells, suggesting KLK4-MCAs are highly invasive in the peritoneal microenvironment. In patient samples, a high level of KLK4 is expressed by ascitic EOC cells compared to matched primary tumour cells, further supporting a role for KLK4 in the ascitic microenvironment. Interestingly, KLK4 transfected SKOV3 cells expressed high levels of urokinase plasminogen activator (uPA), particularly in 3D-suspension, and high levels of both KLK4 and uPA were observed in cells from patient ascites. On the other hand, KLK7-transfected SKOV3 cells also formed large compact MCAs and were resistant to paclitaxel, but high levels of α5/β1 integrin were observed (2), suggesting different signaling pathways induced by these KLKs. In summary, our data suggest that KLK4 inhibition in conjunction with paclitaxel treatment may improve the outcome for women with high KLK4 levels in EOC. Key factors in different signaling pathways induced by KLK4 and KLK7 may also be tested for therapeutic potential for this cancer.