To study the possible correlation between the bioactivity ratios of the serum TSH and serum thyroid hormone concentrations in patients with primary hypothyroidism, the bioactivity of TSH in these patients was determined by means of a sensitive and practical bioassay and the results were compared to those in normal subjects. Sample sera were obtained from 11 patients with primary hypothyroidism and 9 normal subjects. TSH was extracted from the serum with anti-human TSH monoclonal antibody-coated tubes. The bioassay for TSH was performed by measuring the amount of cAMP released into the medium from cultured FRTL-5 cells incubated with the TSH extract. Linear relations were found between the serum TSH values, measured by the immunoassay, and the bioactivity of the TSH, measured by the present assay, in normal subjects (y = 1.13 x-0.3, r = 0.89, p less than 0.01) and in patients with primary hypothyroidism (y = 0.90 x + 0.7, r = 0.93, p less than 0.01). Moreover, the regression line in patients with primary hypothyroidism was not significantly different from that in normal subjects based on the analysis of covariance. The present findings suggest that the bioactivity of TSH is consistent with the immunoreactivity of TSH in patients with primary hypothyroidism as well as in normal subjects.