The TCR beta-chain gene enhancer activates transcription and V(D)J recombination in immature thymocytes. In this paper we present a systematic analysis of the elements that contribute to the activity of the murine TCR beta enhancer in mature and immature T cell lines. We identified a region containing the beta E4, beta E5, and beta E6 motifs as the essential core of the TCR beta enhancer in pro-T cells. In mature cells, the core enhancer had low activity and required, in addition, either 5' or 3' flanking sequences whose functions may be partially overlapping. Mutation of any of the six protein binding sites located within the beta E4-beta E6 elements essentially abolished enhancer activity, indicating that this core enhancer contained no redundant elements. The beta E4 and beta E6 elements contain binding sites for ETS-domain proteins and the core binding factor. The beta E5 element bound two proteins that could be resolved chromatographically and that were both essential for enhancer activity.