Abstract Prostate cancer (PCa) is the second most diagnosed and sixth leading cause of cancer-related death in men worldwide. Activation of β-PDGFR (platelet-derived growth factor receptor) has been demonstrated in 50% of PCa cases and it is part of a 5-gene signature predicting PCa recurrence after prostatectomy. Analysis of the β-PDGFR ligands in PCa revealed association between PDGF D expression and Gleason score as well as tumor stage. When we examined the functional consequences of PDGF ligand-specific β-PDGFR signaling in PCa, we discovered a novel PDGF D-specific autocrine pathway, resulting in activation/shedding of the serine protease matriptase leading to PCa cell invasion, migration, and tumorigenesis. The present study showed that PDGF D, not PDGF B, induces extracellular acidification which correlates with increased matriptase activation. Furthermore, exposure of control or PDGF B expressing cells to an acidic buffer resulted in increased matriptase activation, indicating the functional significance of the acidic milieu in matriptase activation. A cDNA microarray analysis showed that PDGF D/β-PDGFR signaling upregulates expression of the acidosis regulator carbonic anhydrase IX (CA IX), a classic target of the transcriptional factor HIF-1. Interestingly, cellular fractionation displayed a strong HIF-1 nuclear localization in PDGF D expressing cells. Treatment of vector control or PDGF B expressing cells with the HIF-1 activator, CoCl2, specifically led to increased CA IX expression accompanied by extracellular acidosis and matriptase activation. Taken together, these novel findings reveal a new paradigm in matritpase activation involving PDGF D-specific signal transduction leading to extracellular acidosis. Considering the fact that intratumoral acidosis leads to chemo- and radiation resistance, understanding the role PDGF signaling plays in this process will enhance the efficacy of current clinical therapeutics. Citation Format: Abdo J. Najy, Hyeong-Reh C. Kim. The regulation of matriptase activation through PDGF D-mediated extracellular acidosis. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr B55.