The nuclear hormone receptor superfamily currently includes approximately 20 orphan members that do not have known ligands. The prediction that the ligands for these orphans would prove to be important new regulatory compounds has been supported by several recently described orphan - ligand matchups. PPARgamma, for example, has been found to be a receptor for thiazolidinediones, pharmacologically significant antidiabetic agents. We have identified a novel class of ligands for the orphan receptor CAR, which is a constitutive transactivator in the absence of ligand. Strikingly, this constitutive transactivation is shut off by two naturally occurring androstanes, androstanol and androstenol. We propose that these androstanes, or their metabolites, are CAR ligands, and that their mechanism of action is opposite to that of ligands for conventional receptors. Like other nuclear receptor ligands, however, we predict that the CAR ligands will have important biological functions. We propose four specific aims to determine the mechanism of their unexpected inhibitory actions, and to define the functional role of CAR and its ligands. The first aim is to test the specific hypothesis that the CAR AF-2 activation helix is displaced from an active position in the absence of ligand to an inactive position upon ligand binding. The second aim is to screen further androstanes or related compounds for similar inhibitory effects, and to identify true CAR ligands. Since the DNA binding specificity of CAR is very similar to that of the retinoic acid receptor (RAR), the third aim is to test the hypothesis that CAR ligands should affect retinoid signaling pathways. The fourth aim is to determine the consequences of knocking out the CAR gene. Our studies with CAR have already yielded a novel mechanism of action for a nuclear hormone receptor, and we believe that the new information on the actions of its steroid ligands should prove to be equally interesting and important.