Abstract Epithelial Splicing Regulatory Proteins (ESRP1 and ESRP2) have recently been reported as RNA binding proteins that regulate epithelial specific splicing. We identified over-expression of ESRP1 and ESRP2 in ovarian cancer. The aim of this study was to determine the extent of expression of ESRP1 and ESRP2 in ovarian cancers and to examine whether its expression affects the abundance of the epithelial isoform of the Fibroblast Growth Factor receptor 2 (FGFR2) in ovarian cancer. Furthermore, we studied the regulation of FGFR2 splicing following knock-down of ESRPs in ovarian cancer cells. Affymetrix U133 Plus2.0 Array and quantitative real-time polymerase chain reactions (qRT-PCR) were used to measure gene expression levels in ovarian cancers and normal ovaries. ESRP1, ESRP2, epithelial specific FGFR2 IIIb and mesenchymal isoform FGFR2 IIIc mRNA levels were determined via qRT-PCR. RNA interference (RNAi) was used to knock-down ESRP1 and/or ESRP2 to examine its function on determining FGFR2 isoform expression in ovarian cancer cell line OVCAR3. Affymetrix GeneChip expression array analysis of 20 Stage IIIC serous ovarian cancers and 8 normal ovary surface epithelium brushings identified over-expression of ESRP1 (235 fold) and ESRP2 (33 fold). qRT-PCR confirmed that ESRP1 (7586 fold, p < 0.00001) and ESRP2 (21 fold, p <0.00001) were over-expressed in an independent set of 27 ovarian cancers compared to 11 normals. Since ESRP1 and ESRP2 function to drive the epithelial specific splicing of FGFR2, we examined the relative expression of FGFR2 IIIb and FGFR2 IIIc via qRT-PCR. The epithelial specific FGFR2 IIIb was over-expressed (313 fold; p < 0.00001), whereas the mesenchymal isoform FGFR2 IIIc was down-regulated (75 fold). RNAi knock-down of ESRP1 in OVCAR3 cells resulted in increased levels of mesenchymal specific FGFR2 IIIc (6 fold), but had a minimal effect on FGFR2 IIIb expression (−1 fold). ESRP2 knock-down had no detectable effect on FGFR2 IIIc and IIIb expression. However, ESRP1 and ESRP2 double knock-down results in the predominant expression of FGFR2 IIIc (25 fold) and down-regulation of FGFR2 IIIb (−3 fold) when standardized to a scramble control. Our results confirmed that ESRP1, ESRP2 and FGFR2 IIIb are over-expressed and FGFR2 IIIc is down-regulated in ovarian cancers. Also, results of RNAi double knock-down of ESRP1 and ESRP2 indicate that ESRPs drive the expression of the epithelial isoform FGFR2 IIIb in ovarian cancer cells. These data have implications on the ligand specificity of the FGFR2 receptor and for potential individualized therapies targeting FGF signaling in ovarian cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1181.