The transcription factor Pitx3 is expressed selectively in the midbrain and regulates the differentiation and survival of dopaminergic neurons. Lack of this factor results in a degeneration similar to that seen in Parkinson's disease. We have studied the pattern and the level of expression of Pitx3 in dopaminergic neurons of 3- to 4-week-old Wistar rats. We report Pitx3 expression in almost all dopaminergic substantia nigra (SN) and ventral tegmental area (VTA) neurons. It is coexpressed with the neuroprotective marker calbindin (CB) in a larger population of VTA (43%) than SN (16%) dopaminergic neurons. The level of Pitx3 mRNA, determined by semiquantitative RT-PCR, is ≈ 6× higher in VTA than in SN single neurons. In the VTA but not in SN the level of Pitx3 is associated with the presence of CB: in CB-positive neurons the expression of Pitx3 mRNA is 3.6× higher than in CB-negative cells. CB is expressed in a larger population of VTA than SN neurons and the relative level of CB expression is 4× higher in VTA than in SN. A higher Pitx3 expression level and higher coexpression of Pitx3 and CB in VTA than in SN neurons may contribute to the different vulnerability of these dopaminergic nuclei to neurodegeneration.