Selected clones of monoclonal antibodies from mice immunized with solubilized preparations of bovine TSH receptors have been characterized in a cytochemical bioassay (CBA) for thyroid stimulators. This assay is based upon quantification of changes in naphthylamidase activity of sections of guinea pig thyroids with use of a chromogenic substrate. Monoclonal 22A6 is a thyroid-stimulating antibody directed at a site within the TSH receptor. Thus, although it is a weak inhibitor of 125I-TSH binding to thyroid membranes, 22A6 is inhibited from binding to membranes by TSH, exhibits a more than additive agonist effect on adenylate cyclase activity when tested at low TSH concentrations in thyroid cells, and is a competitive antagonist of TSH enhancement of adenylate cyclase activity at high TSH concentrations. In the CBA, 22A6 is a stimulator whose maximal activity is obtained with 77 pg/ml (3-min exposure). Dose-response curves of a long acting thyroid stimulator (LATS)-B standard and 22A6 have slopes which are not significantly different; as anticipated, the response to LATS-B is inhibited by antihuman immunoglobulin G (IgG) and that due to 22A6 by antimouse IgG. In contrast to 22A6, monoclonal 11E8 is a relatively potent inhibitor of 125I-TSH binding as well as TSH stimulation of adenylate cyclase activity, while failing to act as a stimulator itself. 11E8 is itself inactive as a stimulator in the CBA over a wide dose range; it does, however, inhibit TSH stimulation in the CBA. This inhibition is abolished by antimouse IgG. The transient peak of response observed in time courses to TSH occurs later in the presence of 11E8. Unlike its effect on TSH 11E8 shows relatively low potency (greater than 10,000-fold lower) when inhibiting stimulation by the thyroid stimulating antibodies, 22A6 or LATS-B. Since this difference cannot be explained by quantitative differences in the ability of 22A6 or 11E8 to bind to thyroid membranes, the CBA data suggest that the stimulating antibodies, 22A6 and LATS-B, may interact with different determinants on TSH receptors then either TSH or the blocking antibody, 11E8. This also implies that in Graves' disease blocking antibodies may be incompletely expressed in the presence of stimulating antibodies, although they may be potent inhibitors of TSH binding, as measured in receptor assays.