Evaluation of cardiovascular function in mice using micro-CT requires that a contrast agent be administered to differentiate the blood from the myocardium. eXIA 160XL, an aqueous colloidal poly-disperse contrast agent with a high iodine concentration (160 mg I ml−1), creates strong contrast between blood and tissue with a low injection volume. In this study, the blood-pool enhancement time-course of eXIA 160XL is monitored over a 48 h period to determine its optimal use during cardiac function studies in C57BL/6 and BALB/c mice. Eight-second scans were performed (80 kVp, 110 mA) using the GE Locus Ultra micro-CT scanner. Six C57BL/6 and six BALB/c male mice (22–24 g) were injected via tail vein with 5 µl g−1 body weight eXIA 160XL. A precontrast scan was performed; following injection, mice were scanned at 5, 15, 30, 45 and 60 min, and 2, 4, 8, 12, 24 and 48 h. Images were reconstructed, and enhancement–time curves were generated for each of the following tissues: left ventricle (LV), myocardium, liver, spleen, renal cortex, bladder and brown adipose tissue. The highest contrast in the LV occurred at 5 min in both strains (~670 HU above precontrast value). Uptake of the contrast agent by the myocardium was also observed: myocardial tissue showed increasing enhancement over a 4 h period in both strains, remaining even once the contrast was eliminated from the vasculature. In both C57BL/6 and BALB/c strains, eXIA 160XL provided high contrast between blood and myocardial tissue for a period of 30 min following injection. Notably, this contrast agent was also taken up by the myocardium and provided continued enhancement when it was eliminated from the blood, making LV wall motion studies possible. In conclusion, eXIA 160XL, with its high iodine concentration and targeted tissue uptake characteristics, is an ideal agent to use when evaluating cardiovascular function in mice. Copyright © 2012 John Wiley & Sons, Ltd.