We have shown that T. spiralis-specific T lymphocytes can mediate maternal-to-neonatal immunity during lactation. This study addresses the change of lymphocyte populations in rat milk during normal and disease conditions. Two color flow cytometric analysis was performed for milk lymphocytes. T cells (OX19+) made up 45% of rat milk lymphocyte population. T helper cells (Th) composed 35% of total T cells while T cytotoxic/suppressor (Tcs) cells constituted 34%, giving a Th/Tcs ratio of 1.03. The corresponding ratio Th/Tcs in peripheral blood was 2.8. Approximately 21% of OX8+ cells in rat milk were OX19- natural killer (NK) cells. When using the monoclonal antibody 3.2.3 (NKR-P1), 43% of lymphocytes in control rat milk and 14% of blood lymphocytes were NK cells. This indicates a selective passage of these cells into milk. In T. spiralis-immunized rats, the percentage of total T cells was slightly decreased; however, Th and Tcs cells were consistent as compared to control milk. The percentage of NK cells (OX8+OX19-) in milk from T. spiralis-immunized rats was significantly higher than that from control milk (65% vs. 21%, respectively, P less than 0.01). This result was confirmed using the monoclonal antibody 3.2.3 which showed that milk from immunized rats contained 63% NK cells compared to 43% in normal milk (P less than 0.01). This study suggests that NK cells are selectively passaged into rat milk and T. spiralis infection induces an increase of NK cells in milk.