Monolayer cultures of the pigmented epithelial (PE) cells derived from two regions of the pars plicata of bovine eyes were established and grown up to the third passage. After this passage, the cultures became senescent. During the first three passages, the PE cells lost their pigment granules but developed a distinct cellular polarity by forming junctional complexes at their apical cell portions and depositing basement membrane like material on their basal side. The junctional complexes were shown to be impermeable for horseradish peroxidase, suggesting that they contained tight junctions. Histochemically, the monolayer cells stained for carbonic anhydrase (CA) and Na+/K(+)-ATPase, enzymes involved in active fluid secretion. Staining for CA and Na+/K(+)-ATPase as well as for acid phosphatase and immunostaining for vimentin and actin of the cultured PE cells were comparable with that of PE cells in vivo. Therefore, PE monolayer cultures are considered to be a suitable model for experimental studies in vitro.