The relationship between thyroid growth-stimulating antibodies (TGSAb) and thyroid adenylate cyclase-stimulating antibodies (TSAb) in patients with Graves' disease is still a matter of controversy. To investigate this problem, we have developed an assay for the simultaneous measurement of TSAb and TGSAb using FRTL-5 cells. TSAb was detected by its ability to stimulate iodide (I-) uptake and TGSAb by the 3H-thymidine ([3H]-Tdr) incorporation assay. Thirty-four immunoglobulin G (IgG) preparations from patients with active Graves' disease were selected from a previous series in order to include both TSAb-negative IgGs (n = 9) and TSAb-positive IgGs (n = 25) by the cAMP stimulation assay, with a wide range of stimulatory activity. With one exception, the TSAb-positive IgGs produced a significant stimulation of I- uptake; 20 of them were also TGSAb-positive. The nine IgGs negative in the cAMP assay, were also negative in the I-uptake and the [3H]-Tdr incorporation assays. The majority of samples had a similar potency in the two assays and a significant positive correlation was found (r = 0.76; P less than 0.001). Two IgGs previously shown to inhibit TSH-stimulated adenylate cyclase in FRTL-5 cells produced an almost complete inhibition (80-90%) of both TSH- and Graves' IgG-stimulated I- uptake and [3H]-Tdr incorporation. In conclusion, using a simultaneous assay for thyroid growth and adenylate cyclase stimulation, TGSAb in Graves' patients were found only in TSAb-positive IgGs; both Graves' IgG-stimulated activities were inhibited by antibodies blocking the TSH-dependent adenylate cyclase stimulation. Our data strongly suggest that the same antibody may be responsible for both goitre and thyroid hyperfunction of Graves' disease.