To delineate the role of carbohydrate moiety in the expression of in vitro thyrotropic activity of hCG, its variants that lacked sialic acid residues or the entire carbohydrate moiety on one or both subunits were prepared. They along with intact hCG were then tested for the abilities to bind to TSH receptor and stimulate cAMP production and growth responses in FRTL-5 cells. The removal of sialic acid from either one or both subunits sharply increased the [125I]bovine TSH binding-inhibiting activity of hCG in the receptor assay. Among the variants tested, desialylated hCG (as-hCG) was the most potent inhibitor, followed by alpha-as-beta, as-alpha-beta, and hCG in that order. With respect to their abilities to stimulate cAMP generation in the cells, the activities of all desialylated hCG variants were markedly higher than that of hCG itself, and as in the receptor assay, as-hCG was the most potent stimulator tested. At the same concentration (100 micrograms/ml), as-alpha-beta, alpha-as-beta, and hCG were approximately only 57%, 46%, and 27% as active as as-hCG in activating cAMP production. The findings in the growth assay were entirely consistent with those noted in the cAMP response assay. Among the deglycosylated hybrids examined, alpha-dg-beta was the most effective in activating cAMP release. An equivalent dose (200 micrograms/ml) was about 1.7, 2.7, and 3.8 times as active as dg-hCG dg-alpha-beta, and hCG, respectively. The deglycosylated variants of hCG showed a similar pattern of activity in growth response and cAMP accumulation assays. In both cases, alpha-dg-beta was the most potent stimulator, while hCG was the least active. No significant difference between the potencies of dg-alpha-beta and alpha-dg-beta was discerned in the receptor assay; however, deglycosylated hCG (dg-hCG) was sharply more active. Results of these studies strongly suggest that the optimal expression of in vitro thyrotropic activity of hCG variants in FRTL-5 cells may require an alpha-subunit, with intact carbohydrate, in combination with the deglycosylated beta-subunit. Further, they demonstrate that modification of the hCG carbohydrate moiety alone can transform it from a weak agonist to a potent stimulator of in vitro thyrotropic bioactivity. These results also provide further evidence to support the notion that the degree of expression of thyrotropic activity is strongly affected by the species in which the studies are performed.