The current studies were designed to ascertain the fate of hCG bound to rat corpora luteal cell receptors. Graded doses of highly purified hCG (CR119), ranging from 0.1-10.1 micrograms, were injected. Groups of pseudopregnant rats received iodinated hCG, unlabeled hCG, or both. Supraphysiological levels of hCG were used to enhance the internalization of hCG and its receptors. When ovarian membrane pellets (48,000 X g) were subjected to continuous sucrose density ultracentrifugation, two different ovarian membrane fractions (F1 and F2) bound hCG. Although an increase in hCG binding to the F2 membrane fraction was observed between 1-6 h after a single 0.1-microgram [125I]iodo-hCG injection, no subsequent enhanced binding to that fraction was observed. However, the F1 fraction bound at least 3 fold more hCG than did the F2 fraction 1, 6, 12, and 24 h after the injection of 0.1 micrograms [125I]iodo-hCG. When groups of animals were injected with 0.1, 1.0, or 10.0 micrograms unlabeled highly purified hCG, peak serum, ovarian plasma membrane, and ovarian intracellular hCG concentrations were observed at different times after hormone injection and suggested the progressive transfer of hCG from serum to ovarian fractions in a time- and dose-dependent relationship. Although no intracellular hCG was detected until 60 min after the single 0.1-microgram injection of hCG, both serum and membrane-bound levels were measurable within 15 min of that injection. From these observations, we suggest that ovarian intracellular hCG does not reflect significant contamination with serum or interstitial fluid or from significant dissociation of membrane-bound hCG during tissue handling. Finally, when intracellular hCG was subjected to continuous sucrose density gradient ultracentrifugation, a single major 135I peak was observed, and this comigrated with [125I]iodo-hCG. Our interpretation of the foregoing observations is that the major intracellular form of hCG is not receptor bound.