Experimental leishmaniasis is widely used to study the effector functions of T helper cell subsets in vivo. Healing and nonhealing Leishmania major infections have been correlated with T helper 1 and T helper 2 responses, respectively. In the present study, we determined T cell effector functions ex vivo, without any further restimulation and compared them to those obtained following antigen-specific restimulation in vitro. Our results show that T helper cell responses are significantly less polarized when determined ex vivo as compared to those measured after restimulation in vitro. Moreover, the differences in CD4(+) T cell proliferation observed between healer and nonhealer strains of mice differed ex vivo and in vitro. Our results suggest that determination of both ex vivo as well as in vitro T cell responses is crucial to characterize immune responses during experimental leishmaniasis.