Heterogeneity of pituitary TSH was investigated in rats following thyroidectomy. Adult male rats were sacrificed at varying periods (2-28 days) after thyroidectomy. In another experiment, thyroidectomized rats were injected daily with various doses of L-T4 (0.3-7.5 micrograms/100 g body weight, ip) and sacrificed 2 weeks later. The homogenate of the pituitaries was applied on an isoelectric focusing column or a Sephacryl S-200 column. The normal rat pituitary contained 5 major components of immunoreactive (IR) TSH in isoelectric focusing, in which the isoelectric point (pI) ranged from 6.6 to 8.8. The multiple components of IR-TSH beta were observed almost in the same areas as those of IR-TSH. Following thyroidectomy IR-TSH components with more acidic pI, associated with IR-TSH beta, were evident. A large amount of IR-TSH beta in the pituitaries of thyroidectomized rats appeared near the void volume in gel filtration, suggesting the presence of big TSH beta. Supplement of L-T4 minimized these thyroidectomy-induced changes in isoelectric focusing and gel filtration. Furthermore, big IR-TSH beta was little affected by ultracentrifugation and was relatively stable after treatment with 6 M guanidine hydrochloride. We demonstrated that the rat pituitary gland contained multiple components of IR-TSH and IR-TSH beta, both of which became variegated after thyroidectomy. It is suggested that a discernible degree of heterogeneity of TSH, particularly of TSH beta, is dependent upon the increased rate of TSH biosynthesis at the pituitary level.