It has been suggested that replicative senescence might be involved in the pathophysiology of age-related diseases.To study the process of senescence in trabecular meshwork (TM) cells.Porcine TM tissues were obtained and placed in primary cultures with Dulbecco's modified Eagle's medium/Ham's F-12 medium. After 2-3 weeks, migrated and proliferated TM cells were trypsinised and cultured in serial passages, and identified with fluorescein-labelled low-density lipoprotein (DiI-Ac-LDL), a marker of TM cells. Staining for senescence-related beta-galactosidase activity was performed at population doubling level (PDL) 2, 8 and 16 at pH 6. Terminal restriction fragment (TRF) length was examined by Southern blot analysis using a (32)P-labelled telomere-specific sequence (TTAGGG)(3) at each PDL.DiI-Ac-LDL staining revealed that most (nearly 100%) of the cells in the culture were TM cells, which were flattened in shape and positive for senescence-related beta-galactosidase staining at PDL 16. Reduction of TRF length as a function of population doubling was also shown.TM cells exhibited characteristics of senescence at PDL 16 in vitro. The results demonstrated that cellular senescence may be related to the pathophysiology of primary open-angle glaucoma.