The signal responsible for the nuclear localization of the progesterone receptor has been characterized. It is a complex signal. The study of the mechanism of this nuclear localization has revealed that the receptor continuously shuttles between nucleus and the cytoplasm. The receptor diffuses into the cytoplasm and is constantly and actively transported back into the nucleus. The same phenomenon exists for estradiol and glucocorticoid receptors. The mechanism of entry of proteins into the nucleus is well documented, whereas the mechanism of their outward movement to the cytoplasm is not understood. We have grafted different nuclear localization signals (NLSs) onto beta-galactosidase and have studied the traffic of this protein using heterokaryons and microinjection experiments. We have demonstrated that the same NLSs are involved in both the inward and the outward movement of proteins through the nuclear membrane. These results suggest that the nucleocytoplasmic shuttling may be a general phenomenon for nuclear proteins that could possibly undergo modifications in the cytoplasm and exert some biological activities there. These conclusions also imply that at least part of the cellular machinery involved in the nuclear import of proteins may function bidirectionally. Using these techniques, we have shown that the two major antiprogestins, RU486 and ZK98299, act at the same distal level of hormone action.