We have conducted a comprehensive assessment of CCR9 expression and function at the important milestone stages of murine thymocyte development. We reveal an unusually complex regulatory pattern, in which CCR9 influences T cell development at several widely dispersed stages. We find that CCR9 is not expressed within the thymus until the double-negative (DN)3 stage, although it appears to contribute to T cell precursor development prior to residence in the thymus. CCR9 expression is influenced by pre-T cell receptor signals, and is dramatically up-regulated in a population that appears to be transitional between the DN4 and double-positive stages. In the periphery, functional CCR9 is expressed by all naive CD8 T cells, but not by naive CD4 T cells. To our knowledge, this latter finding is the first difference observed in homing receptor expression between naive lymphocyte populations. This suggests that naive CD8 T cells might have access to lymphoid microenvironments from which naive CD4 T cells are excluded.