The catalytic activities of glutathioneS-transferases (GSTs), particularly the α-class isozymes, can provide protection against oxidative stress through GSH-mediated metabolism of reactive products of lipid peroxidation. Lipid peroxidation products from oxidative metabolism in alveolar macrophages play an important role in mediating and regulating inflammatory response and injury in the lung. The rabbit has been used as an important animal model for studies of the role of alveolar macrophages in pulmonary pathology. Although rabbit lung macrophages display GST activity, the isozyme-specific expression of GSTs and the catalytic properties of these isozymes has not previously been defined. In present studies, we have purified the GST isozymes of rabbit alveolar macrophages obtained by bronchoalveolar lavage and performed immunologic and kinetic characterization of the purified isozymes. Results of our studies indicate the presence of three α-class isozymes (pI 10.2, 9.3, and 6.0) and one μ-class isozyme (pI 7.2). N-terminal sequence analysis of the μ-class isozyme indicated that it was distinct from the two previously described μ-class isozymes of rabbit. Kinetic studies indicated that two cationic α-class GSTs (pI 10.2 and 9.3) contribute the large majority of selenium independent GSH-peroxidase activity toward dilinoleoyl phosphatidylcholine hydroperoxide (kcat/Kmvalues of 83.4 and 31.9 s−1· M−1· 103, respectively). A third α-class GST (pI 6.0) was shown to have highest catalytic activity toward conjugation of the 4-hydroxynonenal (4HNE) with GSH (kcat/Km= 1900 s−1· M−1· 103). Structural and immunologic characterization of this GST isozyme indicated that it belongs to a subclass of the α-classGSTs selectively expressed in mesodermal origin cells that are exposed to high levels of oxidative stress and are characterized by high specific activity toward both lipid hydroperoxides and 4-HNE.