ABSTRACT: Mono-(2-ethylhexyl) phthalate (MEHP), the biologically active metabolite of the plasticizer di-(2-ethylhexyl) phthalate, is a member of a class of chemical compounds with known adverse effects on the male reproductive system. Recent studies showed that oxidative stress and mitochondrial dysfunction in germ cells may contribute to phthalate-induced disruption of spermatogenesis. To determine whether the redox-protein mitochondrial thioredoxin-dependent peroxidase, peroxiredoxin 3 (Prx3), may be a component of germ cell homeostasis mechanisms, this study first examined the physiologic relevance of Prx3 in the rodent testis by determining its cell-specific expression. Our findings show that prx3 mRNA is expressed in a developmental, cell-specific manner in rat Leydig cells, Sertoli cells, and germ cells; among mouse germ cells, prx3 expression was highest in spermatocytes, findings consistent with those in rat. In mouse meiotic spermatocytes, Prx3 was strikingly localized at the nuclear perimeter and cytoplasm, findings suggestive of a direct role for Prx3 in determining spermatocyte response to toxicants. To better define the mechanisms involved in male germ cell dysfunction following phthalate exposure, an immortalized mouse spermatocyte-derived germ cell line, GC-2spd(ts), was exposed to MEHP (24 hours; 100 and 200 μM). We determined whether Prx3 and cyclooxygenase-2 (COX-2), pivotal proteins involved in oxidative stress responses in spatially restricted subcellular domains, were affected. Mitochondrial Prx3 and mitochondrial and cytosolic COX-2 significantly increased following 200 μM MEHP treatment; proliferation was inhibited without inducing cell death. Using this germ cell model, the data suggest that changes in cellular oxidation-reduction (redox) homeostasis in the germline can accompany MEHP exposure, disrupting mitochondrial antioxidant defenses, despite absence of phthalate-induced apoptosis.