Abstract Objective Previous studies have revealed a substantial between-centre variability in DCE-MRI biomarkers of hepatocellular function in rats. This study aims to identify the main sources of variability by comparing data measured at different centres and field strengths, at different days in the same subjects, and over the course of several months in the same centre. Materials and methods 13 substudies were conducted across three facilities on two 4.7 T and two 7 T scanners using a 3D spoiled gradient echo acquisition. All substudies included 3–6 male Wistar-Han rats each, either scanned once with vehicle (n = 76) or twice with either vehicle (n = 19) or 10 mg/kg of rifampicin (n = 13) at follow-up. Absolute values, between-centre reproducibility, within-subject repeatability, detection limits, and effect sizes were derived for hepatocellular uptake rate (Ktrans) and biliary excretion rate (kbh). Sources of variability were identified using analysis of variance and stratification by centre, field strength, and time period. Results Data showed significant differences between substudies of 31% for Ktrans (p = 0.013) and 43% for kbh (p < 0.001). Within-subject differences were substantially smaller for kbh (8%) but less so for Ktrans (25%). Rifampicin-induced inhibition was safely above the detection limits, with an effect size of 75 ± 3% in Ktrans and 67 ± 8% in kbh. Most of the variability in individual data was accounted for by between-subject (Ktrans = 23.5%; kbh = 42.5%) and between-centre (Ktrans = 44.9%; kbh = 50.9%) variability, substantially more than the between-day variation (Ktrans = 0.1%; kbh = 5.6%). Significant differences in kbh were found between field strengths at the same centre, between centres at the same field strength, and between repeat experiments over 2 months apart in the same centre. Discussion Between-centre bias caused by factors such as hardware differences, subject preparations, and operator dependence is the main source of variability in DCE-MRI of liver function in rats, closely followed by biological between-subject differences. Future method development should focus on reducing these sources of error to minimise the sample sizes needed to detect more subtle levels of inhibition.