We have investigated the effect of TRH on the accumulation of glycosylated TSH in the rat anterior pituitary gland. Hemipituitaries from adult male rats were incubated in medium containing [3H]glucosamine in the presence of TRH. [3H]Glucosamine-labelled TSH in media and pituitaries was measured by immunoprecipitation and characterized by isoelectric focusing after affinity chromatography. Incorporation of [3H]glucosamine into immunoprecipitable TSH in the media and pituitaries increased progressively with the period of incubation. Although the release of [3H]glucosamine-labelled and unlabelled TSH into media was stimulated by the addition of TRH in a time- and dose-dependent manner, TRH administration did not alter the amounts of labelled or unlabelled TSH in the anterior pituitary lobes. The anterior pituitaries were found, by isoelectric focusing analysis, to be composed of four major component peaks of [3H]glucosamine-labelled TSH. Administration of TRH caused profound changes in the radioactivity of these components and evoked new radioactive peaks, resulting in the appearance of six components in total. The present data provide evidence that TRH significantly changes the heterogeneity of glycosylated TSH in the anterior pituitary without altering the amount of the glycosylated form.