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We investigated the effect of post-labeling delay (PLD) on the evaluation of brain tumor blood flow using arterial spin labeling (ASL) magnetic resonance (MR) imaging to assess the need for imaging with two PLDs. Retrospective analysis was conducted on 63 adult patients with brain tumors who underwent contrast-enhanced MR imaging including ASL imaging with PLDs of both 1525 and 2525 ms on a 1.5 T or 3 T MR unit. Blood flow was estimated in the tumors and normal-appearing brain parenchyma, and tumor blood flow was normalized by parenchymal flow. Estimates of tumor blood flow, parenchymal flow, and normalized tumor flow showed no statistically significant differences between PLDs of 1525 and 2525 ms. Close correlations between different PLDs were found, with the closest correlation for normalized tumor flow. These results were similarly observed for the 1.5 T and 3 T units. The blood flow estimates obtained using ASL MR imaging in patients with brain tumors were highly concordant between PLDs of 1525 and 2525 ms, irrespective of the magnetic field strength. It is indicated that imaging with a single, standard PLD is acceptable for ASL assessment of brain tumor perfusion and that additional imaging with a long PLD is not required.