The objective of this study were to establish a cell line derived from porcine hyalocytes and to investigate the regulation of hyaluronan (HA) synthesis in response to cytokines. After 50 passages of the cells derived from porcine vitreous tissue, a cell line was generated. The immortalized cells showed fibroblastic morphology. The cell doubling time was 56.9h. In the mRNA level, the cells expressed plate-derived growth factor (PDGF) alpha receptor, PDGF beta receptor, transforming growth factor-beta (TGF-beta) type I receptor, TGF-beta type II receptor, CD44, collagen type I, collagen type II, glial fibrillary acidic protein (GFAP), hyaluronan synthase (HAS) 2, HAS 3 and beta-actin. In the protein level, GFAP was expressed in this cell line. S-100 protein and cytokeratin were not detected. Stimulation with TGF-beta1 and/or PDGF-BB induced a marked increase in the expression level of HAS2 mRNA, and induced HA production. TGF-beta1 stimulated HAS2 expression through the signal transduction pathway including Smad 2,3,4. In summary, this report constitutes the first successful immortalization of porcine hyalocyte cells. The production of HA was induced from the generated porcine hyalocyte cell line under the stimulation of TGF-beta1 and/or PDGF-BB, which may be related to the pathogenesis of proliferative membrane formation in proliferative vitreo-retinal diseases.