Hyaluronan synthase 3 (HAS3) is responsible for the production of both secreted and cell-associated forms of hyaluronan and is the most active of the three isoforms of this enzyme in adults. In this study, the cDNA for human HAS3 was cloned and characterized. The open reading frame consisted of 1659 bp coding for 553 amino acids with a deduced molecular weight of about 63,000 and isoelectric pH of 8.70. The sequence of human HAS3 displayed a 53% identity to HAS1 and a 67% identity to HAS2. It also contained a signal peptide and six potential transmembrane domains, suggesting that it was associated with the plasma membrane. To evaluate the physiological role of human HAS3, expression vectors for this protein were transfected into TSU cells (a prostate cancer cell line), and the phenotypic changes in these cells were examined. The enhanced expression of hyaluronan in the transfected cells was demonstrated by dot blot analysis and ELISA. These cells were found to differ from their vector-transfected counterparts with respect to the following: (a) they grew at a faster rate in high (but not low) density cultures; (b) conditioned media from these cells stimulated the proliferation and migration of endothelial cells; (c) when placed on the chorioallantoic membrane of chicken embryos, these cells formed large, dispersed xenografts, whereas the control transfectants formed compact masses; and (d) when injected s.c. into nude mice, the xenografts formed by HAS3 transfectants were bigger than those formed by control transfectants. Histological examination of these xenografts revealed the presence of extracellular hyaluronan that could act as conduits for the diffusion of nutrients. In addition, they had a greater number of blood vessels. However, the HAS3-transfected TSU cells did not display increased metastatic properties as judged by their ability to form lung masses after i.v. injection. These results suggested that the HAS3-induced overexpression of hyaluronan enhanced tumor cell growth, extracellular matrix deposition, and angiogenesis but was not sufficient to induce metastatic behavior in TSU cells.