CD69 is a type II C-type lectin involved in lymphocyte migration and cytokine secretion. CD69 expression represents one of the earliest available indicators of leukocyte activation and its rapid induction occurs through transcriptional activation. In this study we examined the molecular mechanism underlying mouse CD69 gene transcription in vivo in T and B cells. Analysis of the 45-kb region upstream of the CD69 gene revealed evolutionary conservation at the promoter and at four noncoding sequences (CNS) that were called CNS1, CNS2, CNS3, and CNS4. These regions were found to be hypersensitive sites in DNase I digestion experiments, and chromatin immunoprecipitation assays showed specific epigenetic modifications. CNS2 and CNS4 displayed constitutive and inducible enhancer activity in transient transfection assays in T cells. Using a transgenic approach to test CNS function, we found that the CD69 promoter conferred developmentally regulated expression during positive selection of thymocytes but could not support regulated expression in mature lymphocytes. Inclusion of CNS1 and CNS2 caused suppression of CD69 expression, whereas further addition of CNS3 and CNS4 supported developmental-stage and lineage-specific regulation in T cells but not in B cells. We concluded CNS1-4 are important cis-regulatory elements that interact both positively and negatively with the CD69 promoter and that differentially contribute to CD69 expression in T and B cells.