The kinetics of 125I-labeled thyrotropin (125I-TSH) binding to human thyroid receptors are presented. At pH 6.0, binding was maximal (30--35%) and there was one class of binding sites [Kd = 6.8 X 10(-9) M; binding capacity (Ro) = 57 pmol/mg of protein]. At pH 7.4, Scatchard plots of binding were nonlinear, indicating either a single class of negatively cooperative sites (Kd = 3.7 X 10(-9) M; Ro = 26 pmol/mg of protein) or, alternatively, independent high- (Kd = 5.0 X 10(-10) M; Ro = 3 pmol/mg of protein) and low-affinity (Kd = 1.7 X 10(-8) M; Ro = 26 pmol/mg of protein) binding sites. The role of negative cooperativity was evaluated from the rates of association and dissociation at pH 7.4. The kinetically determined binding constants (Kd = 1.7 X 10(-11) M; Ro = 2 pmol/mg of protein) were more similar to those determined for the high-affinity component than to those predicted from the negative cooperativity model. Dissociation of bound TSH was independent of initial site occupancy over a 40-fold range, corresponding to a 100-fold range of free TSH concentration. The dissociation rate of 125I-TSH was enhanced by unlabeled TSH to a similar degree, irrespective of initial binding site occupancy. Because the negative cooperativity model does not accommodate these data, it is concluded that TSH receptors in human thyroid behave kinetically and at equilibrium as a single class of high-affinity sites up to TSH concentrations well above the physiological range.