Following the injection of hepatobiliary contrast agents, MRI detects all molecules included in a region of interest but cannot estimate true concentrations in sinusoids, interstitium, hepatocytes or bile canaliculi. The aim of the study was to measure true concentrations in hepatocytes and to show how transfer rates across sinusoidal and canalicular membranes generate these concentrations. We perfused livers isolated from normal rats with 200 μM Gd-DTPA and Gd-BOPTA and measured clearances from sinusoids to liver and from hepatocytes to bile canaliculi or back to interstitium. We detected Gd-BOPTA with a gamma probe and determined true concentrations in each liver compartment knowing their liver volumes. No pharmacokinetic modelling was applied. Gd-BOPTA clearance from sinusoids to liver (2.5 ± 0.4 mL/min) was 50 times higher than that of Gd-DTPA (0.05 ± 0.02 mL/min) when portal flow rate was 30 mL/min (p < 0.0001). Gd-BOPTA clearance from sinusoids to liver was always superior to hepatocyte clearance, and hepatocyte Gd-BOPTA concentrations measured by the probe increased over time. Gd-BOPTA concentrations reached 439 ± 83 μM in hepatocytes and 15 × 700 ± 3100 μM in bile canaliculi, while concentrations in sinusoids were 200 μM. Gd-BOPTA true concentrations in hepatocytes depend on the simultaneous clearances from sinusoids to hepatocytes and from hepatocytes to bile canaliculi and back to sinusoids. The study better defines how signal intensities are generated when hepatobiliary contrast agents are injected in clinical imaging. Copyright © 2016 John Wiley & Sons, Ltd.