Regional variations in the expression of a striatal enriched protein tyrosine phosphatase called STEP were studied in the adult rat brain by a combination of immunocytochemistry, lesion studies, Western blotting, and in situ hybridization. Monoclonal antibodies generated against STEP identified multiple polypeptides of M(r) 46, 37, 33 and a doublet of M(r) 64–66 kDa on Western blots. Although the three STEP immunoreactive bands with lower molecular weights were enriched in cytosolic fractions, the 64–66 kDa doublet was enriched in membrane fractions. All of the immunoreactive forms were abundant in the caudate-putamen and were present in lower amounts or were undetectable in other brain regions. In substantia nigra, the M(r) 64–66 kDa doublet was not detected but bands with M(r) 46, 37, and 33 kDa were present. Immunocytochemical and lesion experiments demonstrated that the cytosolic STEP isoforms present in the substantia nigra are in presynaptic axons originating from the projection neurons of the caudate putamen, which innervate this structure. Additional in situ hybridization studies showed that STEP mRNA expression patterns correlate with the patterns of immunocytochemical staining. These findings indicate that there are multiple polypeptide isoforms of STEP enriched in the basal ganglia and related structures which differ in terms of their intracellular locations and functional roles.