Accurate prediction of human drug toxicity is a major challenge in drug development. It is estimated that 50% of pre-clinical in-vivo screening of novel drug molecules in rodents fails to predict subsequent human toxicity. Regulatory authorities have recognised the poor performance of current in-vivo screening and have called for the development of in-vitro based screening assays to reduce current animal testing in pre-clinical screening. To address this, we are developing predictive in-vitro primary renal cell models, to replace poorly predictive in-vivo screening studies. Aim: To develop an in-vitro primary renal proximal tubule model from male CD1 mouse kidney. The initial focus was to optimise the isolation protocol, to generate viable cell monolayers of differentiated renal tubule cells and demonstrate the retention of expression and function of ATP-binding cassette (ABC) transport proteins within primary mouse proximal tubule cells. Proximal tubule cells were isolated using a collagenase digest protocol followed by isopycnic centrifugation on a Percoll density gradient. A range of collagenase concentrations were tested to determine the optimum digestion protocol. After 7 days of cell culture, relative mRNA expression levels of ABC transporters were determined using a quantitative RT-PCR array (SABiosciences). To demonstrate functional expression of these transporters, cell monolayers were loaded with either Hoechst 33342 or 5-Chloromethylfluorescein Diacetate (CMFDA) fluorescent dyes in the presence and absence of transporter inhibitors. The highest viable cell yields were found at 0.16 collagenase units per milligram tissue (n=3, P<0.001, t-test). The relative mRNA expression levels of the ABC transporters multidrug resistance protein 1 (ABCC1) and multidrug resistance protein 4 (ABCC4) were higher (2-avgΔCT values of 6.06 & 11.87 respectively) than multidrug resistance protein 3 (ABCC3) (0.70) or multidrug resistance protein 5 (ABCC5) (1.03). We found no expression of multidrug resistance protein 2 (ABCC2) (0.03). Breast cancer resistance protein (ABCG2) and multidrug resistance protein 1b (ABCB1b) expression levels were 0.39 and 0.21 respectively, giving a rank order of ABCC4>ABCC1>>ABCC3>ABCC4>>ABCB1b>ABCG2. Inhibition of Hoechst 33342 efflux from the cell by cyclosporin A (n=3, IC50 3.60±0.10μM, mean ± SEM) and Ko-143 (n=3, IC50 0.52±0.01μM, mean ± SEM) demonstrated expression of multidrug resistance protein 1 (ABCB1) and breast cancer resistance protein (ABCG2) transporters. Inhibition of CMFDA dye efflux by MK-571 (n=3, IC50 2.60±0.06μM, mean ± SEM) demonstrated expression of multidrug resistance protein (ABCC) transporters. This data provides the first steps in the characterisation of a primary renal proximal tubule model from mouse kidney.