Split immunological tolerance refers to states in which an individual is capable of mounting certain types of immune responses to a particular antigenic challenge, but is tolerant of the same antigen in other compartments of the immune system. This concept is applicable to the immunological relationship between mother and fetus, and particularly relevant in equine pregnancy. In pregnant mares, antibody responses to paternal foreign Major Histocompatibility Complex class I antigens are robust, while anti-paternal cytotoxic T cell responses are diminished compared to those mounted by non-pregnant mares. Here, we compared the distribution of the major lymphocyte subsets, the percentage of lymphocytes expressing Interferon Gamma (IFNG) and Interleukin 4 (IL4) and the level of expression of the immunoregulatory transcription factor FOXP3 between pregnant and non-pregnant mares, and between peripheral blood and the endometrium during pregnancy. In a cohort of mares in which peripheral blood lymphocytes were tested during early pregnancy and in the non-pregnant state, there were only slight changes observed during pregnancy. In contrast, comparison of peripheral blood lymphocytes with lymphocytes isolated from the endometrial cups of pregnant mares revealed striking differences in lymphocyte sub-populations. The endometrial cups contained higher numbers of IFNG+ lymphocytes, and lower numbers of lymphocytes expressing IL4. The endometrial cup lymphocytes also had higher numbers of FOXP3+ cells compared to peripheral blood lymphocytes. Taken together, these results strengthen the evidence for a state of split tolerance to trophoblast, and furthermore define sharp differences in immune reactivity during equine pregnancy between peripheral blood lymphocytes and lymphocytes at the maternal-fetal interface.