Confluent monolayers of polygonal epithelioid cells were obtained from enzymatically and mechanically dispersed bovine anterior pituitaries (AP) and pars tuberali (PT). The ultrastructure of the cells composing the monolayer was consistent with the follicular or folliculostellate cells (FC) of the pituitary, i.e., lack of secretory granules; formation of follicles in culture; interdigitations with neighboring cells with numerous tight junctions; presence of extensive microfilaments; and sparse rough endoplasmic reticulum and Golgi apparatus. Culture media from monolayers of first passage cultures contained little if any of the AP hormones' luteinizing hormone, prolactin, and ACTH. Shortly after reaching confluency, regions of the monolayer bulge away from the surface of the culture dish to form domes. Dome formation has been described only with cultures of cells that function as transport epithelia in vivo. FC cultured on polycarbonate filters were placed in Ussing chambers. A transepithelial potential difference of approximately 1.1 mV and a resistance greater than 300 omega cm2 were detectable 4–5 days after plating. The short-circuit current (Isc) was decreased 70% by amiloride applied to the mucosal surface and further decreased by the addition of ouabain at the serosal surface. The beta-adrenergic agonist isoproterenol increased the Isc and this action was prevented by a beta-antagonist. These observations indicate that pituitary FC in culture behave as a transport epithelium. Considering the organization of FC in the AP and PT, they suggest a regulatory role for FC in the maintenance of the ionic composition of the interstitial fluid of the pituitary gland.