The kidney is a key organ in the elimination of drug molecules from the body. Renal drug elimination is mediated by a series of membrane transporters expressed in proximal tubular cells that form effective secretory pathways for drug molecules. Transporter-mediated drug-drug interactions leading to nephrotoxicity is common. Current animal models of renal drug transport are not predictive of human exposure. In parallel to our established human proximal tubule cell model, we have developed primary cultures of rat proximal tubule cells which may allow the comparison of the renal handling of drug molecules in both species. Here we report the characterisation of rat primary proximal tubule cells as a model for drug transporter studies. Tubular cells were isolated from the kidneys from male Sprague-Dawley rats via collagenase digest protocol followed by Percoll gradient centrifugation to purify the proximal tubule cell (PTC). A PCR Transporter array of 86 key transport proteins was used to screen the total cell RNA isolated from cultured rat PTC and native kidney cortical tissue. The results showed Abcc2, Abcc3, Abcc4, Abcc6 and Abcg2 had apparent lower level of expression levels in rat PTC cultured on flasks compared to isolated cells from native tissue. In contrast, Abcb1b expression level was 2-fold higher in cultured cells. The SLC-family members: Slc22a1, Slc22a2, Slc22a6, Slc22a7, Slc16a1 and Slc16a2 were also down-regulated. To measure the functional expression of key efflux transporters, H33342 and CMFDA cellular retention assays were performed. Rat PTC were loaded with H33342 or CMFDA in the presence or absence of CsA, Ko143 and MK-571, inhibitors of Abcb1b, Abcg2 and Abcc1-6 respectively. In the presence of CsA (5µM), cellular retention of H33342 was significantly higher (P<0.001) compared to controls. CsA resulted in a significant reduction in the concentration of H33342 that produced half maximal fluorescence (Km) from 3.44±1.10μM to 0.14±0.03μM, a result consistent with inhibition of transporter-mediated dye efflux. The presence of Ko143 (1µM) also increased cellular retention of H33342 (P<0.01) with a significant change in Km for dye loading from 3.44±1.10μM to 0.22±0.08μM. Similarly, retention of CMFDA metabolite GSMF in the presence of Mrp inhibitor MK-571 (10µM) was significantly higher than control. To recapitulate monolayer formation, rat PTC were grown on Transwell filter supports and the transepithelial electrical resistance (TEER) measured. The TEER of the cells after 2 days of growth increased by 2-fold compared to baseline (P<0.05) and by day 4, TEER peaked at 4.5 times the base resistance (P<0.001). These data suggest that rat cells form confluent cell monolayers on permeable filter supports. In summary, these results show rat PTC have the potential to be used as an important in vitro model for drug transporter and drug-drug interaction studies.