In the majority of radioimmunoassays (RIA) reported for TSH in human serum, detectable levels (average 4–5 μU/ml) have been found in normal subjects and in patients with thyrotoxicosis. In an attempt to define these two groups more clearly, the specificity of the TSH RIA was increased by incubating assay standards (MRC Research Standard A) in hormone-free human serum, and sensitivity was improved by the use of low concentration of tracer (25 pg) and dilute antiserum (1:300,000). Assay sensitivity ranged from 0.2–0.8 μU/ml serum (mean 0.4). The following results were obtained: euthyroid subjects (69), mean 1.0 (range <0.4–3.1) μU/ml; thyrotoxic patients (43) undetectable in 36, 0.2–1.6 in remainder; hypothyroid (76) mean 111 (range 5.6–704). Triiodothyronine treatment resulted in undetectable TSH levels in 17/22 subjects tested. Conclusion: improvements in specificity and sensitivity of the RIA resulted in normal values separable from those in thyrotoxicosis and in good agreement with the reported biological assay findings on a large pool of normal serum. These improvements should allow better definition of thyroid pathophysiology, particularly after treatment of thyrotoxicosis.