Thyrotoxicosis in Graves' disease is often aggravated in early pregnancy and is closely associated with postpartum recurrence of stimulative thyrotoxicosis. To examine whether thyroid-stimulating TSH receptor antibody (TSAb) or human chorionic gonadotropin (hCG), which also has thyroid-stimulating activity (TSA), was responsible for this early aggravation, the respective TSA due to TSAb or hCG was evaluated by a highly sensitive cAMP accumulation assay using FRTL-5 cells. TSA was detectable in all of 11 women in normal early pregnancy, correlated positively with serum hCG concentration, and was abolished completely by the pretreatment of serum sample with the solid-phase hCG antibody coupled with Sepharose 4B. The model serum samples of Graves' disease with pregnancy were made by the mixture of normal pregnant and Graves' sera, and their TSA were reduced by the pretreatment with the solid-phase hCG antibody, just corresponding with the reduction in hCG-induced TSA. TSA of early pregnant sera in 20 patients with Graves' disease decreased significantly but were still positive even after the pretreatment with the hCG antibody. Serial changes in TSAb and hCG-induced TSA were measured in 5 of these 20 pregnant patients. hCG-induced TSA increased associated with the increase in free thyroxine, while TSAb did not show striking change in early pregnancy. These data indicate that (1) respective TSA due to TSAb or hCG can be measured distinctively by using the solid-phase hCG antibody and (2) hCG plays a crucial role in the aggravation of Graves' thyrotoxicosis in early pregnancy.