Fluctuations in blood-oxygenation level dependent (BOLD) signal and perfusion affect the quantification of changes in cerebral blood flow (CBF), coupled to neuronal activity, in arterial spin labeling (ASL). Subtraction methods for control and labeled MR images (i.e. pair-wise, surround subtraction, and subtraction of sinc-interpolated images), postulated to mitigate this interference in pseudo-continuous ASL (pCASL), were evaluated by comparison with quantitative (15)O-water PET. At rest, a good agreement in the CBF values was found between PET and MRI for each of the subtraction methods. Stimulation of the visual system resulted in a regional CBF increase in the occipital lobe, which was detectable in both modalities. Bland-Altman analysis showed a systematic underestimation of the CBF values during activation in MRI. Evaluation of the relative CBF change induced by neuronal stimulation showed good inter-modality agreement for the three subtraction methods. Perfusion data obtained with each subtraction method followed the stimulation paradigm without significant differences in the correlation patterns or in the time lag between stimulation and perfusion response. Comparison to the gold standard confirmed the detectability of a neuronal stimulation pattern by pCASL. The results indicate that the combined use of background suppression and short TE reduces the BOLD-weighting in the pCASL signal.