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.