To determine whether incubation of mouse thyrotropic tissue with TRH in vitro influenced the oligosaccharide structure of TSH, thyrotropic tumor tissue or pituitary tissue was incubated in vitro with [3H]mannose or with [35S]sulfate and [3H]methionine, in the absence or presence of TRH for times up to 24 h. [3H]mannose-labeled oligosaccharides from intracellular TSH and free alpha-subunits were analyzed by paper chromatography, and were predominantly Man9GlcNAc and Man8GlcNAc units both in the absence and presence of TRH. The [35S]sulfate/[3H]methionine ratio in secreted molecules was greater for TSH than for free alpha-subunits; within TSH heterodimers the ratio was greater for beta-subunits than alpha-subunits. The [35S]/[3H] ratio was not altered in TSH or free alpha-subunits by TRH. Analyses of [3H]mannose-labeled charged oligosaccharides by HPLC anion-exchange chromatography revealed similar types of oligosaccharides present on TSH subunits and free alpha-subunits (having one or two sulfate residues, one or two sialic acid residues, or both a sulfate and a sialic acid residue). These charged oligosaccharides occurred in different proportions on TSH subunits compared to free alpha-subunits, and also differed depending on whether the tissue source was tumorous or nontumorous. The proportions of oligosaccharide unit types were not altered by TRH. Thus, while this study provided information concerning the high-mannose and complex oligosaccharides of mouse TSH, there was no evidence that short incubations of tissues with TRH in vitro caused modulation of TSH oligosaccharide structures.