The role of CD44 in T cell biology remains incompletely understood. Although studies using anti-CD44 antibodies have implicated this cell adhesion molecule in a variety of important T cell processes, few T cell defects have been reported in CD44-deficient mice. We have assessed the requirement for CD44 in T cell development and mature T cell function by analyzing mice in which CD44(-/-) and WT cells were produced simultaneously. In mixed (CD44(-/-) + CD44(+/+)) bone marrow chimeras, production of CD44(-/-) T cells was shown to be reduced compared to WT cells due to inefficient intrathymic development. In addition, mature CD44(-/-) CD8(+) T cells generated a substantially lower response than WT T cells after infection of mice with lymphocytic choriomeningitis virus, with the reduction in response apparent in both lymphoid and non-lymphoid tissues. Overall, these results demonstrate a poor capacity of CD44(-/-) T lineage cells to compete with WT cells at multiple levels, implicating CD44 in normal T cell function.