Purpose. The present study was conducted to develop and characterize a functional primary culture of pigmented rabbit conjunctival epithelial cells on permeable support exhibiting tight barrier properties.Methods. Conjunctival epithelial cells were isolated by 0.2% protease treatment, cultured at 0.5–1.8×106cells/cm2onto collagen-treated Transwell filters, and were maintained either in the presence of 1 % fetal bovine serum throughout or serum-free media from day 3 onwards. Transepithelial potential difference (PD) and transepithelial electrical resistance (TEER) were measured and equivalent short-circuit current (Ieq= PD/ TEER) estimated.Results. There appears to be a critical plating density of 1.5×106cells/cm2for functional development of tight epithelial cell cultures. The culture conditions as noted above did not affect either the time when peak bioelectric parameters were attained (days 8–10) or the magnitude of these parameters at a plating density of 1.5×106cells/cm2. Specifically, cells grown in a serum-free media showed a peak TEER of 1.9±0.2 kÒ.cm2, a PD of 14.2±1.6 mV (apical side negative), and an Ieqof 8.0±0.4μA/cm2(mean±SEM, n = 45). Electron microscopy of serum-weaned cultures revealed a multilayered epithelium with numerous microvilli on the outermost layer of cells, while sporadic positive Periodic Acid Schiff (PAS) staining under light microscopy suggested the presence of mucin-secretory goblet cells.Conclusions. A functional, tight, epithelial barrier of the pigmented rabbit conjunctiva on a permeable support has been developed, which may be useful for mechanistic studies of ion and drug transport at the cellular level.