Glycosylation of newly synthesized subunits of TSH, a regulator of thyroid hormone synthesis and secretion, was studied in a mouse pituitary thyrotropic tumor in vitro. Subunits were pulse labeled by incubations of the tumor for 10 or 30 min with [35S]methionine, followed by chase incubations with unlabeled methionine for 30--90 min. The sensitivity of the subunit oligosaccharides to cleavage by the glycosidase endo-beta-N-acetylglucosaminidase H (Endo H) was determined by comparisons of the electrophoretic mobilities, on sodium dodecyl sulfate-polyacrylamide gels, of Endo H-treated and untreated labeled subunits in cells and media. We found that oligosaccharides of most of the intracellular subunits remained sensitive, whereas the oligosaccharides of all subunits released from the tumor were resistant to cleavage by Endo H during the transport of the subunits within the secretory pathway. Therefore, alterations in the carbohydrate structure of the subunits occur close to the time of secretion of the subunits and presumably represent late distal glycosylation steps that convert the oligosaccharides from high mannose to complex types. Late glycosylation may serve extraglandular functions, such as enhanced metabolic stability or expression of recognition sites for interaction with target organ receptors.