The interstitial nucleus of the posterior limb of the anterior commissure (IPAC) is exclusively innervated by tyrosine hydroxylase-immunoreactive (TH-IR) fibers as observed in the other nuclei of the rat forebrain such as the striatum and nucleus accumbens. Distinguishing TH-IR afferents to the IPAC from those projecting to neighboring nuclei has been difficult. However, we previously showed that the TH-IR fibers projecting to the IPAC were invulnerable to neurodegeneration in zitter mutant rats, whereas almost all TH-IR afferents fibers to the dorsolateral striatum were lost, indicating that these two groups of TH-IR afferents have distinct neurochemical properties. Here, to explore this observation further, we injected Fluorogold (FG) retrograde tracers to identify neurons projecting to the IPAC or dorsal striatum. We also determined the distribution of attractin mRNA and protein, causative factors for the pathological phenotypes of zitter mutant rats, within the normal rat midbrain. In rats injected with FG into the dorsal striatum, we detected many FG-positive neurons in the ventral aspect of the substantia nigra pars compacta (SNC). In contrast, many FG-positive neurons were observed in the dorsal aspect of the SNC of rats injected with FG into the IPAC. Immunohistochemistry and in situ hybridization studies of intact animals revealed that both attractin mRNA and protein were expressed at higher levels in the ventral aspect of the SNC, whereas both attractin mRNA and protein were expressed at lower levels in the dorsal aspect of the SNC. Taken together, these results indicate that TH-IR afferents to the IPAC have distinct neurochemical properties from those to the striatum and may account for the differential vulnerability to neurodegeneration observed in zitter mutant rats.