hCG is a known stimulator of mouse thyroid in vivo. Studies were therefore performed to ascertain whether the thyroid-stimulating activity of hCG in the mouse could also be demonstrated by the in vitro techniques that had failed to show any activity of hCG in the human thyroid. When labeled with 125I and incubated at 22°C in 20 mM Tris-0.5% bovine serum albumin (Tris-BSA), pH 7.45, with increasing concentrations (70–300 μg protein/ml) of a mouse thyroid fraction, a purified hCG preparation ([ 125I]hCG) showed 5–12% specific binding. In contrast, its binding to a human thyroid particulate fraction, over the same range of protein concentrations, did not exceed 1%. When similar studies were performed at 37°C in 10 mM Tris-50 mM NaCl-0.5% BSA, pH 7.45, [ 125I]hCG showed no detectable binding either to the human or the mouse thyroid fractions. At concentrations ranging from 1 to 20 mIU/ml (0.9–18 × 10 −9 M), bTSH stimulated cAMP release from human thyroid slices into the medium in a dose-dependent manner. In contrast, hCG concentrations from 10 3 to 10 4 IU/ml (2–20 × 10 −6 M) were without effect on cAMP release. bTSH, at concentrations of 4.5 and 9.0 mIU/ml (4 and 8 × 10 −9 M), stimulated cAMP release from the mouse thyroid, producing in the medium approximately 11- and 28-fold increases in cAMP concentration. hCG also stimulated cAMP release from the mouse thyroid, the increases being approximately 2.3- and 1.8-fold, in the presence of 2270 and 4540 IU/ml (4.5 and 9.0 × 10 −6 M), respectively. Our data suggest that sensitivity of the thyroid to stimulation by hCG varies in different species, ranging from relatively high in the mouse to little or none in the human.