Swimbladder gas gland cells are polar epithelial cells which release acidic metabolites through the membranes of an extensive basolateral labyrinth, and secret surfactant via exocytosis at apical membranes. We have developed a method to establish primary cell cultures of gas gland cells in order to establish a model system for physiological analysis of gas gland cell function in vitro. Isolated gas gland cells attach to collagen S coated surfaces. Cells cultured in collagen S coated petri dishes were flat and showed no histological polarity. Cells cultured on Anodisc membranes in a superfusion system, in which the apical and basal side of the cells was supplied with a saline solution and with glucose containing DMEM cell culture medium, respectively, showed a clear polarity similar to the in vivo situation. Measurement of lactate release at the apical side and at the basal side revealed that these cells were functionally polar and secreted at least 70% of the lactate at their basal membranes. Gas gland cells could also be cultured in an air/liquid system, in which the apical membrane was exposed to humidified air. Cells cultured under these conditions released lactate only on the basal side and histologically were similar to cells cultured in the superfusion system.