We have determined the structures of high mannose (Man) oligosaccharide units of TSH, free alpha-subunit, and non-TSH-related total glycoproteins (TP) within microsomal subfractions of mouse thyrotropic tumor. Tumor minces were incubated with D-[2-3H]Man, homogenized, and subfractionated into rough endoplasmic reticulum (RER) as well as proximal and distal smooth endoplasmic reticulum/Golgi apparatus. TSH subunits and TP were precipitated from these fractions, and high Man units released by endoglycosidase H were analyzed by paper chromatography. Glc3Man9GlcNAc2 (Glc = glucose; GlcNAc = N-acetylglucosamine) was not detected in TSH subunit precursors in any fraction, but was detected in TP. Glc1Man9GlcNAc2 accumulated in TSH with chase, but only small amounts were detected in free alpha-subunit and TP. Trimming of Man9GlcNAc2 to Man8GlcNAc2 began in RER before 1 h for all species, but the rate of Man trimming was rapid for free alpha-subunit, moderate for TSH, and slow for TP. Man8GlcNAc2 was a rate-limiting step in processing for all species in the RER. Man5GlcNAc2 was a second major rate-limiting step in processing of free alpha-subunits only and accumulated in distal smooth endoplasmic reticulum/Golgi apparatus. Other studies in progress suggest that a function of the thyrotroph is to modulate the structure of TSH oligosaccharide units during biosynthesis to achieve mature hormone with physiologically appropriate metabolic clearance and intrinsic biological activity. The present study supports this notion, since we found that the qualitative nature and kinetics of processing differ for oligosaccharide units of TSH, free alpha-subunit and TP within the microsomes.