Using a clonal strain of cultured Leydig tumor cells (designated MA-10), we have compared the fate of the receptor-bound ovine LH (oLH) and human CG (hCG) in cells incubated in the presence or absence of extracellular Na+. We have previously shown that Na+ does not affect the number of LH/CG receptors or the binding affinity of hCG, but it decreases the binding affinity of oLH. Thus, it was possible to compare the fate of these two hormones under conditions where their binding affinities differ by a factor of 8 (i.e. in the presence of Na+) or by a factor of less than 2 (i.e. in the absence of Na+). Moreover, since only the affinity of oLH is affected by Na+, we were able to distinguish between those effects mediated by a change in binding affinity from those effects that are more general in nature by comparing the behavior of hCG in cells incubated in the presence or absence of Na+. The results presented herein show that the rates of internalization of oLH and hCG are very similar regardless of the presence or absence of Na+; and the absence of Na+ leads to a 2- to 3-fold decrease in the rate of degradation of the internalized oLH and hCG. We have found, however, that the binding affinities of oLH and hCG have significant effects on the pathway of receptor-mediated endocytosis under conditions where there is no free hormone present in the medium. The results presented show that in the absence of free hormone in the medium, the rate of hormone internalization can be approximated from the rate of disappearance of the surface-bound hormone only if the binding affinity of the hormone is high enough so that there is little or no dissociation of the hormone from the receptor during the course of the experiment (i.e. hCG in the presence or absence of Na+, but oLH only in the absence of Na+). If the binding affinity of the hormone is low (i.e. oLH in the presence of Na+), then the rate of disappearance of the surface-bound hormone represents the sum of the rates of internalization and dissociation of the hormone and thus cannot be used to approximate the rate of hormone internalization.