Habituation is a basic process of learning in which repeated exposure to a sensory stimulus leads to a decrease in the strength of neuronal activations and behavioral responses. In addition to increases in neuronal activity, sensory stimuli can also lead to decreases in neuronal activity. Until now, the effects of habituation on stimulus-induced neuronal deactivations have not been investigated. We performed functional magnetic resonance imaging in 30 healthy subjects during repetitive unilateral somatosensory stimulation and combined this analysis with a psychophysiological examination of changes in the perception threshold. Consistent with the literature, we found a time-dependent decrease of the positive blood oxygenation level-dependent (BOLD) response (indicative of habituation) in the primary somatosensory cortex (SI) contralateral to the stimulus. In contrast, the negative BOLD response (NBR) in the ipsilateral SI did not show a decrease in amplitude; instead, an increase in amplitude was found, i.e., a stronger NBR (increased response). The increased NBR was associated with an increased perception threshold of the nonstimulated hand. These findings suggest that habituation is not primarily characterized by a decrease in the neuronal response to repeated stimuli but rather a widespread change in the balance between excitatory and inhibitory effects that favors inhibitory effects. Hum Brain Mapp 35:152–160, 2014. © 2012 Wiley-Periodicals, Inc.