We investigated at which point during thymocyte differentiation functions were acquired that are characteristic for mature Th cells. Differentiation from CD3+CD69-, CD4+CD8+ double-positive (DP) cells to terminally differentiated CD3+, CD4+CD8- single-positive (SP) cells was broken down into six discrete stages that were purified by four-color sorting: CD69-CD3+DP (stage 0), CD69+CD27-DP (stage 1), CD69+CD27-CD4+SP (stage 2), CD27+CD1+CD4+SP (stage 3), CD1-CD45RO+CD4+SP (stage 4), and CD1-CD45RO-CD4+SP cells (stage 5). Phenotypically, these stages seem to describe consecutive steps in differentiation from immature stage 0 to the terminally matured stage 5. Functionally, the capacity to proliferate on IL-2 after stimulation was absent in CD69- stage 0 cells, but was acquired gradually during stages 1 to 4. Clonal expandability and the capacity to respond to stimulation with the production of cytokines were acquired later and rather abruptly by CD1- stage 4 and 5 cells. Activation markers such as CD69 expression and in vivo IL-2 gene transcription came up simultaneously at the DP stage and peaked at stage 3 to 4. These data suggest that functional maturation of Th cells occurs over an extended period in differentiation, stages 1 to 4, and coincides with a gradual increase in activation markers. After completion of functional differentiation, at stage 5, in vivo IL-2 mRNA transcription and CD69 expression are down-regulated, and the cells become functionally resting naive T cells expressing CD45RA+.