The influence of 5,5'-diphenylhydantoin (DPH) on specific T3 binding by rat hepatic nuclear T3 receptors was determined in tissue obtained from euthyroid and athyreotic rats. Nuclear binding of [125I]T3 was determined in isolated liver nuclei 60 min after the injection of DPH (20 mg/100 g BW) or vehicle (V). Mean specific nuclear T3 binding was decreased 14% (P less than 0.01) and 24% (P less than 0.02) from the control value in two experiments with nuclei from euthyroid rats and 23% (P less than 0.02) in nuclei from athyreotic rats. These findings in athyreotic rats and the observation that endogenous nuclear T3 was not increased in euthyroid rats after DPH injection suggested that the observed decrease in specific T3 binding after DPH injection did not result from displacement of endogenous T3 from plasma proteins or other binding sites into the nucleus. Further studies employing isolated nuclei revealed that the DPH-induced decrease in specific nuclear T3 binding appeared independent of duration of incubation up to 90 min and dose related. Maximum inhibition of specific T3 binding was 14.0% (P less than 0.001) and occurred in incubates that contained 320 microM DPH. Moreover, this DPH effect was evident throughout the full range of receptor saturation by T3. Similar effects of DPH were observed in studies using solubilized nuclear T3 receptors. A mean 14.0% decrement (P less than 0.001) in specific T3 binding was observed throughout the 72-h time course of these studies. Moreover, the DPH-induced changes occurred when solubilized receptors were up to 62% saturated with T3. These studies show that DPH injection or addition in vitro results in a 10-20% decrease in specific T3 binding by isolated hepatic nuclei or solubilized hepatic nuclear T3 receptors. This effect of DPH may result from a relatively weak interaction at nuclear sites and the very high concentration of DPH compared to T3 that prevails in DPH-treated animals.