We previously established that two subcutaneous injections of viable syngeneic testicular germ cells (TGC) alone can induce CD4+ T cell-dependent experimental autoimmune orchitis (EAO) in mice. This model is histologically characterized by lymphocytic infiltration into the testes and the following aspermatogenesis. In the present study, we investigated the dynamics of splenic cytokines in EAO, using specific enzyme-linked immunosorbent assay. We found that splenic production of both type 1 helper T cell (Th1) and type 2 helper T cell (Th2)-related cytokines increased after the second but not the first immunization with TGC, indicating that secondary immune responses are critical to the EAO induction. However, the production of Th1-related cytokines became predominant at the clinical stage of EAO. Additionally, serum FSH and inhibin-B increased and decreased, respectively, during EAO. On the other hand, LH did not significantly change and testosterone temporally increased during the same period. These results indicate that both Th1- and Th2-related cytokines are involved at the pre-clinical phase of EAO, but that Th1- rather than Th2-related cytokines are responsible for the clinical phase when spermatogenesis is disrupted but the Leydig cell function is well preserved.