Thymocytes mature in response to the cues from the thymic micro-environment, which regulate stage-specific gene expression during development. We find that several proteins that bind the kappa B sequence in vitro are constitutively activated in freshly isolated thymocytes. These include the rel-related p50 homodimers, p50/p65 heterodimers, low levels of c-rel, and two other factors that may be thymus specific. Disruption of the thymic micro-environment resulted in loss of DNA-binding, suggesting that lymphocyte-stromal cell interactions induce and maintain these proteins in a DNA-binding form. Phorbol ester and ionomycin treatment induced p50, p65, and p68 c-rel kappa B DNA-binding activity. Expression of p68 c-rel protein, but not p50 or p65, was suppressed by the immunosuppressive drug FK506. Because FK506 specifically inhibits the appearance of mature single-positive thymocytes, gene expression regulated by p68 c-rel may play a role in selection and maturational signals involved in the double-positive to single-positive transition.