We previously reported that mice implanted with mammary tumors show a progressive thymic involution that parallels the growth of the tumor. The involution is associated with a severe depletion of CD4+8+ thymocytes. We have investigated three possible mechanisms leading to this thymic atrophy: 1) increased apoptosis, 2) decreased proliferation, and 3) disruption of normal thymic maturation. The levels of thymic apoptosis were determined by propidium iodide and annexin V staining. A statistically significant, but minor, increase in thymic apoptosis in tumor-bearing mice was detected with propidium iodide and annexin V staining. The levels of proliferation were assessed by in vivo labeling with 5′-bromo-2′-deoxyuridine (BrdU). The percentages of total thymocytes labeled 1 day following BrdU injection were similar in control and tumor-bearing mice. Moreover, the percentages of CD4−8− thymocytes that incorporated BrdU during a short term pulse (5 h) of BrdU were similar. Lastly, thymic maturation was evaluated by examining CD44 and CD25 expression among CD4−8− thymocytes. The percentage of CD44+ cells increased, while the percentage of CD25+ cells decreased among CD4−8− thymocytes from tumor-bearing vs control animals. Together, these findings suggest that the thymic hypocellularity seen in mammary tumor bearers is not due to a decreased level of proliferation, but, rather, to an arrest at an early stage of thymic differentiation along with a moderate increase in apoptosis.