Dynamic contrast-enhanced MRI is often used to assess the response to therapy in small animal models of cancer. Rigorous quantification of dynamic contrast-enhanced MRI data using common pharmacokinetic models requires dynamic determination of the concentration of contrast in tumor tissue and in blood. Measurement of the blood concentration, or vascular input function (VIF), requires high temporal resolution and is prone to distortion as a result of flow and partial volume artifacts when measured in local blood vessels. We have developed a strategy for the robust measurement of VIF in mice that uses a constrained reconstruction algorithm to enable sampling from the left ventricle of the heart. The feasibility of the algorithm and its resistance to cardiac motion are demonstrated in vivo and through numerical simulations. VIF sampling is interleaved with slices dedicated to tumor coverage to yield a fast VIF sampling period (81 ms) that is decoupled from the temporal resolution of tumor data (3.9 s). The algorithm provides results that agree with fully encoded measurements in the slowly varying component of VIF to within a 4% root-mean-square signal difference. Analysis of a parametric representation of VIFs measured in a population of mice showed a significant reduction in variations observed within subjects (5%–58% over four parameters; p < 0.05) and a reduction in variations between subjects (19%–62%) when using this technique. Preliminary dynamic measurements in an orthotopic xenograft model of anaplastic thyroid cancer revealed a decrease in the variation of pharmacokinetic parameters between mice by a factor of two. Copyright © 2010 John Wiley & Sons, Ltd.