BACKGROUND AND PURPOSE: The arterial input function (AIF) is critical in determining hemodynamic parameters quantitatively with bolus-tracking MRI. We studied the effect of varying the location of measurement of AIF on the volume of hypoperfusion. We compared the volumes of hypoperfusion obtained with different AIFs with the final ischemic lesion volume. METHODS: We included 13 patients with acute cerebral ischemia in the anterior circulation who underwent diffusion- (DWI) and perfusion (PWI)-weighted imaging within 8 hours after symptom onset and exhibited DWI lesion expansion between baseline and follow-up. AIF was measured at 4 locations: near both middle cerebral arteries (MCAs), in MCA branches adjacent to the largest DWI abnormality, and at the same level on the opposite hemisphere. Hypoperfusion lesion volumes were compared with the DWI volume at follow-up. RESULTS: Large variations in PWI lesion size were found with different AIF locations. The largest PWI lesions were found when AIF was measured at the contralateral MCA. Smaller PWI lesions were found when AIF was measured in the other locations. There was no significant difference between PWI lesion area at baseline and follow-up DWI lesion when AIF was measured at the contralateral MCA. The other PWI lesions significantly underestimated follow-up DWI lesion size. CONCLUSIONS: AIF is an important determinant of the size of hypoperfusion lesions measured with PWI. PWI lesion volumes determined with AIF from the contralateral MCA are associated with follow-up lesion volume.