The thyroid-stimulating antibody (TSAb) of Graves' disease is an IgG; its immunochemical characteristics were analyzed using an increase in the concentration of cyclic AMP in the human thyroid slice in vitro for its assay. IgG from serum known to contain TSAb in high concentration was purified and H chains from this IgG were combined with Bence Jones k or lambda L chains or original L chains; reconstitution of IgG was confirmed by agar gel and immunoelectrophoresis. In studies with 6 sera there was no thyroid stimulation on testing such recombined molecules except for minimal activity in one instance of recombination of original H and L chains. In 8 other experiments TSAb-IgG containing only k or lambda L chain was isolated by affinity chromatography. In 7 instances thyroid-stimulating potency was entirely with IgG lambda, except for minor activity in IgGk in 3 of these studies; in the eighth preparation TSAb was predominantly in IgGk with minimal activity in IgG lambda that was assayed at a much higher concentration. By chromatography on Protein A Sepharose, IgG3 was obtained and it was found to contain no TSAb; activity was, however, recovered in the Protein A-retained fraction (ie IgG 1+ 2+ 4). With 3 preparations of TSAb-IgG lambda, subclasses 1+ 2+ 4, removal by affinity chromatography of subclass 4 led to full recovery of TSAb in subclasses 1+ 2. On subsequent removal of subclass 2, in two instances all activity was retained by subclass 1; in the third preparation some TSAb was not accounted for. These data, together with previous reports from this laboratory showing a relatively constant pI for TSAb-IgG on preparative isoelectric focusing, are compatible with its having restricted heterogeneity, instead of being polyclonal as described by others.