Background: Cell-mediated immunity is critical for controlling infection and preventing reactivation during the chronic phase of Toxoplasma gondii infection. In people suffering from AIDS, T. gondii is one of the major opportunistic infectious agents. Mechanisms regulating rapid development of clinical signs in previously asymptomatic patients remain unclear; however, cofactors such as stress are suspected to play a role in the susceptibility to opportunistic infections. Objective: This study examined the role of cold stress (CS) in splenocyte function during chronic T. gondii infection. Methods: Control mice and mice previously infected orally with T. gondii were subjected to CS during the chronic phase (CSchr), i.e. 90 days after infection, and in vitro cell proliferation and cytokine production were measured before (day 0) and 1, 15 and 25 days after CSchr. Splenocyte proliferation and cytokine production were measured after in vitro stimulation with concanavalin A (Con-A), anti-CD3 antibody (A-CD3) and Toxoplasma lysate antigen. Results: CSchr enhanced splenocyte proliferation in cells stimulated with Con-A and A-CD3, but it suppressed proliferation in cells stimulated with T. gondii antigens. Increased levels of interferon (IFN)-γ were detected independent of the type of stimulation after CSchr and remained high throughout the experiment. CS had similar results in noninfected animals. Conclusion: Although an overall increase in splenocyte function occurred after nonspecific stimulation, CS suppressed primed spleen cells from responding to T. gondii antigens which could lead to reactivation of latent infection. The increase in IFN-γ after CSchr could be a result of spleen cells being primed by released parasites by this stressor. IFN-γ is critical in the control of parasite reactivation.