Uncertainty in arterial input function (AIF) estimation is one of the major errors in the quantification of dynamic contrast-enhanced MRI. A blind source separation algorithm was proposed to determine the AIF by selecting the voxel time course with maximum purity, which represents a minimal contamination from partial volume effects. Simulations were performed to assess the partial volume effect on the purity of AIF, the estimation accuracy of the AIF, and the influence of purity on the derived kinetic parameters. In vivo data were acquired from six patients with hypopharyngeal cancer and eight rats with brain tumor. Results showed that in simulation the AIF with the highest purity is closest to the true AIF. In patients, the manually selection had reduced purity, which could lead to underestimations of Ktrans and Ve and an overestimation of Vp when compared with those obtained by the proposed blind source separation algorithm. The derived kinetic parameters in the tumor were more susceptible to the changes in purity when compared with those in the muscle. The animal experiment demonstrated good reproducibility in blind source separation-AIF derived parameters. In conclusion, the blind source separation method is feasible and reproducible to identify the voxel with the tracer concentration time course closest to the true AIF. Magn Reson Med, 2012. © 2012 Wiley Periodicals, Inc.