Ovarian cancer metastasis progresses through a series of stages, each of which occurs in a distinct microenvironment. Notably, metastasising ovarian cancer cells, shed from the primary ovarian tumour, form multicellular structures (spheroids) within the peritoneal fluid, which contribute to tumour dissemination.We have previously shown that the type III TGFβ receptor (TGFBR3, or betaglycan) is lost from the surface of the majority of metastatic granulosa cell tumours (GCTs) and blocks migration and invasion of GCT cell lines in monolayer cultures (Bilandzic et al., 2009, Mol Endocrinol 23:539-548). In the current work, we utilised the KGN GCT cell line and three-dimensional non-adherent spheroid culture to test the hypothesis that TGFBR3 blocks GCT metastasis at the spheroid stage. This approach demonstrated that TGFBR3-expressing spheroids form more slowly than control spheroids when grown suspended in methylcellulose (2 d vs 1 d). Furthermore, using the xCELLigence Real-Time Cell Analyzer, we show that, compared to control spheroids, TGFBR3-expressing spheroids exhibit decreased attachment to fibronectin and collagen IV, two matrix components of the peritoneum. Furthermore, while control spheroids rapidly disaggregated overnight following replating onto plastic or glass, TGFBR3-expressing spheroids exhibited no disaggregation during the same time period. Finally, TGFBR3-expressing spheroids were 2-fold less invasive through Matrigel or a monolayer of human stromal cells (p<0.05). Western blot analysis of total cell lysates revealed that MMP2, MMP15, MMP16, and N-cadherin, markers of motile, invasive ovarian cancer cells, were significantly downregulated in the TGFBR3-expressing cells compared to control cells. Collectively, our data indicate that loss of TGFBR3 during ovarian cancer progression contributes to intraperitoneal metastasis by enhancing spheroid formation and invasion of the peritoneum. Conversely, retention of TGFBR3 by GCT cells may hamper tumour dissemination by an MMP-dependent mechanism. Supported by the NHMRC of Australia (RegKeys 494802; 441101; 388904) and Victorian Government Infrastructure funds.