Stress-induced alterations in expression of c-fos protein (Fos) in mesencephalic dopamine (DA) neurons of the rat were examined in order to discern which midbrain DA neurons are metabolically activated by stress. Restraint stress for 30 min increased the number of DA neurons exhibiting Fos-like immunoreactivity in the ventral tegmental area (VTA), but not in the substantia nigra or retrorubral field. Stress elicited an increase in the number of DA neurons expressing Fos in specific nuclei within the VTA. Administration of the anxiogenic beta-carboline FG 7142 also increased the total number of VTA DA neurons expressing Fos protein, whereas pretreatment with an anxiolytic benzodiazepine (diazepam) partially prevented the stress-induced increase in Fos expression. Restraint stress for 30 min increased concentrations of the DA metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) in the nucleus accumbens and striatum, as well as in the prefrontal cortex. Retrograde tracer studies revealed that stress increased Fos protein expression in a distinct subset of DA neurons projecting to the prefrontal cortex. In contrast, Fos expression was not increased in any DA neurons projecting to the nucleus accumbens. The present data indicate that there are at least two functionally distinct DA systems embedded within the prefrontal cortex of the rat.