The in vitro stability of the Ah receptor from rat hepatic cytosol was evaluated by [ 3H]TCDD binding studies, gel filtration, and sucrose density gradient ultracentrifugation. Thermal inactivation of unoccupied receptor followed first-order kinetics between 5 and 40 °C, with an estimated E a for inactivation of ~35 kcal/mol. Protease inhibitors did not reduce and dilution slightly increased the inactivation rate at 20 °C. Recovery and 20 °C stability decreased with increasing ionic strength. The TCDD-receptor complex was less susceptible to degradation at 20 °C, even in the presence of 0.4 m KCl. Specific binding was markedly pH dependent, with maximum recovery at 7.6. Analysis of the pH curve suggested that cysteine sulfhydryl groups may be involved in TCDD binding. Dithiothreitol (1 m m) maximized recovery and 20 °C stability, and addition of the thiol largely reactivated binding sites lost from cytosol prepared without it. Removal of low molecular weight components of cytosol by gel filtration resulted in a rapid 20 °C inactivation rate that could not be lessened by dithiothreitol. Glycerol (10% v v ) and EDTA (1.5 m m) maximized recovery of specific binding, but both decreased 20 °C stability in a concentration-dependent manner. Calcium chloride (4 m m) increased stability at 20 °C by ~20%, and retarded the characteristic shift in sedimentation coefficient from ~9 to ~6 S in high-salt sucrose gradients. The fact that sodium molybdate (20 m m) decreased recovery and 20 °C stability when dithiothreitol was present but slightly increased stability in its absence suggested an antagonism between the two compounds. Molybdate mitigated the inactivation induced by 0.4 m KCl, an effect which may be related to the observation of dual peaks in molybdate-containing high-salt sucrose gradients. These data indicate that (i) thermal inactivation of the unoccupied rat hepatic Ah receptor primarily may be due to physical rather than enzymatic processes; (ii) sulfhydryl oxidation, removal of low molecular weight cytosolic components, and high ionic strength result in rapid rates of inactivation at 20 °C; and (iii) the large degree of protection conferred by TCDD binding implies a very tight ligand-receptor interaction, and as such accords with TCDDs extraordinary potency and persistence in producing its toxic and biochemical effects.