The aim of our study was to develop a magnetic resonance (MR)-compatible in vitro model containing freshly isolated rat hepatocytes to study the transport of hepatobiliary contrast agents (CA) by MR imaging (MRI). We set up a perfusion system including a perfusion circuit, a heating device, an oxygenator, and a hollow fiber bioreactor (HFB). The role of the porosity and surface of the hollow fiber (HF) as well as the perfusate flow rate applied on the diffusion of CAs and O2 was determined. Hepatocytes were isolated and injected in the extracapillary space of the HFB (4 x 10(7) cells/mL). The hepatocyte HFB was perfused with an extracellular CA, gadopentetate dimeglumine (Gd-DTPA), and gadobenate dimeglumine (Gd-BOPTA), which also enters into hepatocytes. The HFB was imaged in the MR room using a dynamic T1-weighed sequence. No adsorption of CAs was detected in the perfusion system without hepatocytes. The use of a membrane with a high porosity (0.5 microm) and surface (420 cm2), and a high flow rate perfusion (100 mL/min) resulted in a rapid filling of the HFB with CAs. The cellular viability of hepatocytes in the HFB was greater than 85% and the O2 consumption was maintained over the experimental period. The kinetics of MR signal intensity (SI) clearly showed the different behavior of Gd-BOPTA that enters into hepatocytes and Gd-DTPA that remains extracellular. Thus, these results show that our newly developed in vitro model is an interesting tool to investigate the transport kinetics of hepatobiliary CAs by measuring the MR SI over time.