ABSTRACT Studying changes in brain activation according to the valence of emotion-inducing stimuli is essential in the study of emotions. Due to the ecological potential of virtual reality, it is also important to examine whether brain activation in response to emotional stimuli can be modulated by the three-dimensional (3D) properties of the images. Using functional Magnetic Resonance Imaging, this study compared differences between 3D and standard (2D) visual stimuli in the activation of emotion-related brain areas. The stimuli were organized in three virtual-reality scenarios, each with a different emotional valence (pleasant, unpleasant and neutral). The scenarios were presented in a pseudo-randomized order in the two visualization modes to twelve healthy males. Data were analyzed through a GLM-based fixed effects procedure. Unpleasant and neutral stimuli activated the right amygdala more strongly when presented in 3D than in 2D. These results suggest that 3D stimuli, when used as "building blocks" for virtual environments, can induce increased emotional loading, as shown here through neuroimaging.