Dopamine appears critical in regulating spatial working memory (SWM) within the PFC of non-human primates; however findings in humans are less clear. Recent studies of the effects of global depletion of dopamine via acute tyrosine/phenylalanine depletion (TPD) on SWM task performance have yielded inconsistent results, which may be partly related to task differences. These previous studies do not address whether TPD can directly impair PFC functioning. The current study investigated the effects of TPD on (1) regional cerebral blood flow (rCBF) during a SWM n-back task using H(2) (15)O Positron Emission Tomography (PET), and (2) behavioural performance on three different SWM tasks. Ten healthy males were scanned twice: once following a placebo (balanced) amino acid mixture and once following an equivalent mixture deficient in tyrosine/phenylalanine (TPD condition). Participants completed two additional delayed-response tasks to examine whether differences in response demands influenced TPD effects on performance. TPD resulted in widespread increases in rCBF, with maximum increases in the region of the parahippocampal gyrus bilaterally, left inferior frontal gyrus, and the putamen. TPD related rCBF reductions were observed in the medial frontal gyrus bilaterally, right inferior temporal gyrus and the pons. Despite widespread changes in blood flow following TPD, no specific effects on SWM neural networks or task performance were observed. The use of three different SWM tasks suggests that task differences are unlikely to account for the lack of effects observed. These findings question the capacity of TPD to consistently modulate dopamine function and SWM neural networks in humans.