Renal proximal tubule cells from the winter flounder (Pseudopleuronectes americanus) were maintained in a functionally differentiated state for up to 16 days in primary culture on floating collagen gels. The cells were confluent after 7-8 days in culture, contracted the collagen gels, and exhibited ciliary activity. Electron microscopy indicated that the cultures were composed of continuous sheets of columnar epithelial cells that had established structural polarity. When mounted in Ussing chambers, the cultures exhibited a small mucosa-negative potential difference (0.6 +/- 0.10 mV) and a low transepithelial resistance (23 +/- 2.3 omega X cm2). Short-circuit current averaged 24 microA/cm2. The cultured epithelium was four times more permeable to Na than to Cl and actively secreted sulfate and p-aminohippuric acid and reabsorbed hexoses. Glucose reabsorption was rheogenic and occurred via a high-affinity (Km = 0.16 mM), low-capacity (Vmax = 5 microA/cm2), phlorizin-sensitive transport system. We concluded that the cultured cells express many of the differentiated properties of the intact flounder proximal tubule and thus provide a suitable model system for studying renal transport processes.