All glycoprotein hormone subunit preparations possess low biological activity when subjected to conventional bioassays. To determine whether the subunits of hCG and LH possess intrinsic biological activity, a variety of hCG and bovine or ovine LH subunits of known biological activity in vivo were assayed in vitro for target cell binding and steroidogenic activity in the rat testis. Binding-inhibition activity in the rat testis homogenate-125I-hCG radioligand-receptor assay system, and steroidogenic activity in the isolated rat testis, were found in all cases to be of similar magnitude to the biological activity of subunits measured in vivo by conventional bioassays. Because enhanced binding-inhibition or steroidogenic properties of isolated subunits were not demonstrable during the appropriate in vitro studies, it was concluded that the individual subunits do not possess preferential binding or activation functions in the dissociated state. In addition, the close agreement between the results of bioassay and in vitro assays indicated that the low biological activity of glycoprotein hormone subunits is most likely to be caused by residual intact hormone in the subunit preparations.