The genes encoding effector molecules of mature T cells, IL-2, perforin and IL-4, were found to be expressed in vivo in the most primitive subsets of thymocytes of adult mice. These subsets have previously been identified by their cell surface markers and by their expression of other T lineage-associated genes. While IL-2, perforin and IL-4 are expressed in distinct patterns, all three are expressed before the induction of RAG-1 and pre-TCR alpha mRNA expression, and are confined to subsets of cells that apparently have not yet undergone commitment to the T lineage. Thus, expression of T cell response genes appears to be one of the earliest markers of lymphocyte differentiation. Activation events marked by CD69 induction occur in these early cell types, but the response gene expression by these cells is separable from CD69 expression. IL-2 and perforin are induced again much later in thymocyte development, during TCR-dependent repertoire selection. At those stages, IL-2 protein and RNA levels per cell are higher, but the fraction of cells expressing IL-2 appears to be much lower than in the most immature stages. In addition, a striking feature of the immature populations is the robust IL-2 expression by presumptive immature NK cells. These findings are discussed in terms of the developmental origins of lineage specificity in T cell response gene regulation.