Neonatal thyrotoxicosis induced by transferred TSH receptor antibody (TRAb) is the ideal human in-vivo experimental system for the evaluation of TRAb. The clinical significance of circulating TRAb in Graves' disease was evaluated by this 'natural in-vivo human assay'. TRAb activity in vitro was measured by radioreceptor assay (thyrotrophin-binding inhibitor immunoglobulin, TBII) and sensitive cAMP accumulation assay using FRTL-5 cells (thyroid-stimulating antibody, TSAb). Further, the binding-stimulation index (B-S index) was newly introduced, which was the most useful indicator for prediction of neonatal thyrotoxicosis, calculated as the product of TBII and TSAb (Tamaki et al., 1988a). Maternal serum TRAb indices showed highly significant correlations with the serum free T4 index (FT4I) and free T3 index (FT3I) in neonates (5-10 days after birth) born to 20 mothers with Graves' disease who had positive TBII and/or TSAb (FT4I: r = 0.825 for TBII, r = 0.908 for TSAb, r = 0.944 for the B-S index, P less than 0.001; FT3I: r = 0.622 for TBII, P less than 0.01, r = 0.812 for TSAb, r = 0.791 for the B-S index, P less than 0.001; n = 20). In contrast, in 57 untreated adult patients with hyperthyroid Graves' disease, the FT4I and FT3I levels were not correlated with any of the TRAb indices. The linear regression relationship between the B-S index and FT4I found in neonates was applied to values in adult patients with Graves' disease, and the patients were divided into three groups on the basis of the 95% confidence limit: high, normal, and low responders of thyroid hormone (FT4I) secretion to the B-S index. FT4I and the ratio of FT4I to the B-S index were highest and the TRAb indices were lowest in the high responders, while FT4I and the FT4I/B-S index ratio were lowest and the TRAb indices were highest in the low responders. The FT4I/B-S index ratio was inversely correlated with the titres of antithyroid microsomal antibody in all the adult patients with untreated Graves' disease (r = -0.288, P less than 0.05). The results suggest that in-vitro assays using animal thyroid cells and cAMP as an index of response are suitable for detecting circulating thyroid stimulating activity in vivo. Secretion of thyroid hormones in Graves' disease may be regulated not only by circulating thyroid-stimulating antibodies but also by intrathyroidal stimulatory factors or by inhibitory or destructive factors.