Catechol-O-methyltransferase (COMT) has been shown to be critical for prefrontal dopamine flux, prefrontal cortex-dependent cognition and activation. Several potentially functional variants in the gene have been identified, but considerable controversy exists regarding the contribution of individual alleles and haplotypes to risk for schizophrenia, partly because clinical phenotypes are ill-defined and preclinical studies are limited by lack of adequate models. Here, we propose a neuroimaging approach to overcome these limitations by characterizing the functional impact of ambiguous haplotypes on a neural system-level intermediate phenotype in humans. Studying 126 healthy control subjects during a working-memory paradigm, we find that a previously described risk variant in a functional Val158Met (rs4680) polymorphism interacts with a P2 promoter region SNP (rs2097603) and an SNP in the 3′ region (rs165599) in predicting inefficient prefrontal working memory response. We report evidence that the nonlinear response of prefrontal neurons to dopaminergic stimulation is a neural mechanism underlying these nonadditive genetic effects. This work provides an in vivo approach to functional validation in brain of the biological impact of complex genetic variations within a gene that may be critical for its clinical association.