Several variables in the reagents and procedures used in the TSH [thyrotropin] radioreceptor assay, the binding of iodinated TSH to its thyroidal receptor. Iodinated bovine TSH (S.A. [specific activity] 30 U[units]/mg) was more effectively bound to receptor than iodinated human TSH (S.A. 7.3 U/mg). Iodination of TSH with the Bolton-Hunter acylation method apparently prevented binding to TSH receptor. Surgically removed human thyroid tissue specifically bound 10.3 .+-. 1.0 (mean .+-. SEM [standard error of the mean]) of added [125I]TSH, but post-mortem human thyroid bound only 3.9 .+-. 0.4% of [125I]TSH (P < 0.001). Maximal binding of [125I]TSH was found at pH 5.8. Many tissue preparations contained activity, possibly due to proteases, which inactivated TSH, and inclusion of a protease inhibitor, aprotinin, significantly increased specific binding.