Essential fatty acids play a crucial role in the activity of several neurotransmission systems, especially in the monoaminergic systems involved in cognitive and motor aspects of behavior. The present study investigated whether essential fatty acid dietary restriction over two generations could differentially affect dopaminergic cell populations located in the substantia nigra rostro-dorso-medial (SNrm) or caudo-ventro-lateral (SNcv) regions which display distinct neurochemical profile and vulnerability to lesions under selected pathological conditions. Wistar rats were raised from conception on control or experimental diets containing adequate or reduced levels of linoleic and α-linolenic fatty acids, respectively. Stereological methods were used to estimate both the number and soma size of tyrosine hydroxylase (TH)-immunoreactive neurons in the SNrm and SNcv. TH protein levels were assessed with Western blots. Long-term treatment with the experimental diet modified the fatty acid profile of midbrain phospholipids and significantly decreased TH protein levels in the ventral midbrain (3 fold), the number of TH-positive cells in the SNrm (∼20%) and the soma size of these neurons in both SNrm (∼20%) and SNcv (∼10%). The results demonstrate for the first time a differential sensitivity of two substantia nigra dopaminergic cell populations to unbalanced levels of essential fatty acids, indicating a higher vulnerability of SNrm to the harmful effects induced by docosahexaenoic acid brain deficiency.