In multimeric glycoproteins, like glycoprotein hormones, mutual subunit interactions are required for correct folding, assembly and transport in the secretory pathway. However, character and time course of these interactions need further elucidation. The influence of the glycoprotein hormone -subunit (GPH) on the folding of the human chorionic gonadotropin -subunit (hCG) in hCG -heterodimers was investigated in [³⁵S]Met/Cys labeled JEG-3 cells. Completeness of disulfide bridge formation during the time-course of folding was estimated by labeling with [³H]N-ethylmaleinimide of free thiol groups not yet consumed. Subunit association took place between immature hCG (high ³H/³⁵S ratio) and almost completely folded GPH. Analysis revealed a highly dynamic maturation process comprising of at least 8 main hCG folding intermediates (molecular weights from 107 kDa to 28 kDa) that could be micro-preparatively isolated and characterized. These hCG variants developed while being associated to GPH. The 107 kDa variant was identified as a complex with calnexin. In contrast to hCG -heterodimers, free non-associated hCG, free large GPH, and GPH homodimers showed a fast-track-like processing in the secretory pathway. At 10 min before hCG secretion sialylation of these variants had already been completed in the late Golgi, whereas hCG -heterodimers had still not arrived medial Golgi. This shows that the GPH in the hCG -heterodimers decelerates the maturation of the hCG portion in the heterodimer complex. This results in a postponed approval of hCG -heterodimers by the endoplasmic reticulum (ER) quality control unlike GPH homodimers, free hCG, and GPH subunits.