We have reported in the preceding paper that human hepatoma (Hep G2) cells synthesize thyroxine-binding globulin (TBG). In this paper, we evaluated the kinetics of secretion of the protein and the effects produced by the ionophore monensin and the glycosylation inhibitor tunicamycin. Cells were pulse labeled with [35S]methionine and then chased after addition of excess unlabeled methionine. TBG appeared in the medium after 10 min, and 50% of the protein was secreted after 45 min. After 2 h, more than 85% of TBG had been released. The rate of secretion of TBG was much slower than that of albumin, 50% of which was secreted after 20 min. Monensin, 1 microM, caused a marked delay in TBG secretion, with 50% released after 80 min. After 2 h, less than 60% had been released and a plateau was approached. Endoglycosidase H (endo H) treatment of intracellular and secreted TBG showed no alteration in the rate of conversion of TBG oligosaccharide units from high-mannose type (endo H-sensitive) to complex type (endo H-resistant), thus suggesting that monensin impeded the exit of TBG from the Golgi apparatus without affecting the terminal glycosylation of the protein. Tunicamycin, 5 micrograms/ml, completely blocked glycosylation and markedly affected TBG secretion, almost doubling the time required for the secretion of 50% of the protein. The effect was specific for TBG, since it was not observed in the case of albumin. After 2 h, only 56% of the protein had been released. Analysis of intracellular and extracellular immunoprecipitated products revealed the presence of aggregates (Mr greater than 100,000). The lack of carbohydrates, although not preventing TBG secretion, had marked quantitative effects, and increased the susceptibility to aggregation.