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Arterial transit time (ATT), a key parameter required to calculate absolute cerebral blood flow in arterial spin labeling (ASL), is subject to much uncertainty. In this study, ASL ATTs were estimated on a per‐voxel basis using data measured by both ASL and positron emission tomography in the same subjects. The mean ATT increased by 260 ± 20 (standard error of the mean) ms when the imaging slab shifted downwards by 54 mm, and increased from 630 ± 30 to 1220 ± 30 ms for the first slice, with an increase of 610 ± 20 ms over a four‐slice slab when the gap between the imaging and labeling slab increased from 20 to 74 mm. When the per‐slice ATTs were employed in ASL cerebral blood flow quantification and the in‐slice ATT variations ignored, regional cerebral blood flow could be significantly different from the positron emission tomography measures. ATT also decreased with focal activation by the same …