The expression of recombination activating gene (RAG) products, responsible for T cell receptor (TcR) gene rearrangement, is shut off during positive selection of thymocytes. The precise stage at which this down-regulation occurs remains somewhat controversial. We have analyzed RAG-1 expression in thymocytes of TcR transgenic mice carried on selecting versus non-selecting genetic backgrounds, both by in situ hybridization on thymus sections and by polymerase chain reaction amplification of RNA from sorted cells. The data from several transgenic lines indicate that RAG expression is already reduced in immature, cortical, CD4+CD8+ cells in the presence of positively selecting major histocompatibility complex molecules, although complete shut-off is not achieved until the mature, medullary, single-positive stage. This finding has practical and theoretical significance for studies on the mechanism of positive selection.