The human LH of the anterior pituitary is a member of the glycoprotein hormone family that includes FSH, TSH, and placental CG. All are noncovalently bound heterodimers that share a common α-subunit and β-subunits that confer biological specificity. LHβ and CGβ share more than 80% amino acid sequence identity; however, in transfected Chinese hamster ovary (CHO) cells, LHβ assembles with the α-subunit more slowly than does hCGβ, and only a fraction of the LHβ synthesized is secreted, whereas CGβ is secreted efficiently. To understand why the assembly and secretion of these related β-subunits differ, we studied the folding of LHβ in CHO cells transfected with either the LHβ gene alone, or in cells cotransfected with the gene expressing the common α-subunit, and compared our findings to those previously seen for CG. We found that the rate of conversion of the earliest detectable folding intermediate of LH, pβ1, to the second major folding form, pβ2, did not differ significantly from the pβ1-to-pβ2 conversion of CGβ, suggesting that variations between the intracellular fates of the two β-subunits cannot be explained by differences in the rates of their early folding steps. Rather, we discovered that unlike CGβ, where the folding to pβ2 results in an assembly-competent product, apparently greater than 90% of the LH pβ2 recovered from LHβ-transfected CHO cells was assembly incompetent, accounting for inefficient LHβ assembly with the α-subunit. Using the formation of disulfide (S-S) bonds as an index, we observed that, in contrast to CGβ, all 12 LHβ cysteine residues formed S-S linkages as soon as pβ2 was detected. Attempts to facilitate LH assembly with protein disulfide isomerase in vitro using LH pβ2 and excess urinary α-subunit as substrate were unsuccessful, although protein disulfide isomerase did facilitate CG assembly in this assay. Moreover, unlike CGβ, LHβ homodimers were recovered from transfected CHO cells. Taken together, these data suggest that differences seen in the rate and extent of LH assembly and secretion, as compared to those of CG, reflect conformational differences between the folding intermediates of the respective β-subunits.