Ketamine acts as an N-methyl-D-aspartate receptor antagonist and evokes psychotomimetic symptoms resembling schizophrenia in healthy humans. Imaging markers of acute ketamine challenge have the potential to provide a powerful assay of novel therapies for psychiatric illness, although to date this assay has not been fully validated in humans. Pharmacological magnetic resonance imaging (phMRI) was conducted in a randomized, placebo-controlled crossover design in healthy volunteers. The study comprised a control and three ketamine infusion sessions, two of which included pretreatment with lamotrigine or risperidone, compounds hypothesized to reduce ketamine-induced glutamate release. The modulation of the ketamine phMRI response was investigated using univariate analysis of prespecified regions and a novel application of multivariate analysis across the whole-brain response. Lamotrigine and risperidone resulted in widespread attenuation of the ketamine-induced increases in signal, including the frontal and thalamic regions. A contrasting effect across both pretreatments was observed only in the subgenual prefrontal cortex, in which ketamine produced a reduction in signal. Multivariate techniques proved successful in both classifying ketamine from placebo (100%) and identifying the probability of scans belonging to the ketamine class (ketamine pretreated with placebo: 0.89). Following pretreatment, these predictive probabilities were reduced to 0.58 and 0.49 for lamotrigine and risperidone, respectively. We have provided clear demonstration of a ketamine phMRI response and its attenuation with both lamotrigine and risperidone. The analytical methodology used could be readily applied to investigate the mechanistic action of novel compounds relevant for psychiatric disorders such as schizophrenia and depression.