Protein-tyrosine phosphatases (PTPases) play a key role in the regulation of insulin action. In order to identify PTPases in skeletal muscle, the major site of insulin-mediated glucose disposal in vivo, we purified PTPases from rat muscle tissue fractions by a series of column chromatographic techniques. PTPase activities were assayed by measuring the dephosphorylation of a rat insulin receptor kinase domain, derivatized lysozyme and p-nitrophenylphosphate, and the enzymes were further characterized by immunoblotting. Of the total PTPase activity in muscle homogenates, 51-64% was localized to the solubilized particulate fraction, with the specific PTPase activity 3.3-fold and 5.6-fold higher in the particulate fraction towards RCM-lysozyme or the insulin receptor, respectively. The major peak (> 75%) of PTPase activity in the particulate fraction was purified further to 700-fold; 75% of this activity passed through a Blue-3GA column and revealed immunoreactivity for both LAR and SH-PTP2. PTPase activity retained on the Blue-3GA column contained PTPase1B. The major peak (> 70%) from muscle cytosol was further purified to 1500-fold. After the Blue-3GA step, immunoblotting revealed both SH-PTP2 and PTPase1B in the cytosol fraction, but LAR was absent from this fraction. LRP (RPTP-alpha) was not detected by blotting the PTPase activities from the purified particulate or cytosol fractions. Immunodepletion studies demonstrated that LAR, SH-PTP2 and PTPase1B were quantitatively major PTPase activities in the initial muscle homogenate, together accounting for over 70% of the total activity towards RCM-lysozyme. These studies provide insight into the relative abundance and subcellular distribution of specific PTPases in muscle tissue that are involved in the regulation of reversible tyrosine phosphorylation in this tissue.