The ventral tegmental area (VTA), primary source of the mesocorticolimbic dopaminergic system, is regarded as a critical site for initiation of behavioural sensitization to psychostimulants. The present study was undertaken to identify the neural pathways converging on the VTA that are potentially implicated in this process. Rats were sensitized by a single exposure to amphetamine (5 mg/kg, s.c.). The distribution of VTA-projecting neurons activated by amphetamine was examined by combining retrograde transport of the cholera toxin β subunit (CTb), injected into the VTA, with immunodetection of Fos. The quantitative analysis of CTb–Fos double labelling demonstrates that amphetamine induced a rapid activation of Fos in a large number of brain areas projecting to the VTA. More than half of the CTb–Fos double-labelled neurons were located in the prefrontal cortex, lateral preoptic area–lateral hypothalamus, pontomesencephalic tegmentum, dorsal raphe nucleus, ventral pallidum and nucleus accumbens. In addition, scattered CTb–Fos double-labelled cells were observed in many other VTA afferent structures, such as claustrum, lateral septum, diagonal band–magnocellular preoptic nucleus, deep mesencephalic nucleus, oral part of pontine reticular nucleus and dorsomedial tegmental area. This suggests that systemic amphetamine activates a wide population of neurons projecting to the VTA that may be important for the modulation of neurobehavioural plasticity produced by this psychostimulant.