Suboptimal performance in working memory (WM) tasks and inefficient prefrontal cortex functioning are related to dysregulation of dopaminergic (DA) and hypothalamic-pituitary-adrenal systems. The aim of the present study was to investigate the joint effect of genetic polymorphisms coding for DA catabolism and glucocorticoid receptor (GR, NR3C1) on brain functioning. The study group (90 right-handed white Caucasian healthy individuals) underwent functional magnetic resonance imaging experiments to examine blood oxygenation level dependent (BOLD) response during a WM task with varying cognitive load (1-, 2- and 3-back). We have also examined skin conductance response (SCR) during the WM task and resting-state cerebral blood flow with continuous arterial spin labelling. The genetic markers of interest included Catechol-O-Methyl-Transferase (COMT) (Met(158)Val) and NR3C1 single-nucleotide polymorphisms (BclI C/G rs41423247, 9β A/G rs6198 and rs1866388 A/G). Haplotype-based analyses showed (i) a significant effect of COMT polymorphism on left anterior cingulate cortex, with greater deactivation in Met carriers than in Val/Val homozygotes; (ii) a significant effect of BclI polymorphism on right dorsolateral prefrontal cortex (DLPFC), with greater activation in G/G carriers than in C carriers and (iii) an interactive effect of BclI (G/G) and COMT (Met/Met) polymorphisms, which was associated with greater activation in right DLPFC. These effects remained significant after controlling for whole-brain resting-state blood flow. SCR amplitude was positively correlated with right DLPFC activation during WM. This study demonstrated that GR and COMT markers exert their separate, as well as interactive, effects on DLPFC function. Epistasis of COMT and BclI minor alleles is associated with higher activation, suggesting lower efficiency, of DLPFC during WM.Molecular Psychiatry advance online publication, 15 November 2011; doi:10.1038/mp.2011.145.