Epithelial cell lines from the proximal tubule of SHR and WKY rats were generated by microdissection, cell growth on 3T3 cell feeder layers, and transduction of the SV40 large T-antigen gene. The cell lines that formed confluent, electrically-resistive monolayers (basal conductance 1 to 20 mS/cm2) were selected for further study. Of these, cell lines generated from one rat did not show evidence of T-antigen expression or integration, and apparently immortalized spontaneously. Cell lines from three other rats expressed high levels of T-antigen, and showed evidence of integration of one or more copies of T-antigen. All cell lines formed polarized monolayers with apical microvilli, tight junctional complexes, and convolutions of the basolateral plasma membrane. Most cell lines grew in the absence of extracellular glucose indicating a capacity for gluconeogenesis. Sodium succinate cotransport and P2-purinergic receptor mediated signaling were demonstrated in all lines tested. The cell lines also showed that Na/H exchanger activity is regulated by angiotensin II. The results indicate that these cell lines express a proximal tubular phenotype, and are morphologically and functionally similar to primary cultures. These rat cell lines represent a new, potentially useful cell model for elucidating the cellular and molecular mechanisms of genetic differences in proximal tubule Na+ reabsorption.