A new and simple in vivo bioassay suitable for routine testing of pituitary and recombinant TSH preparations was developed. Male Albino Swiss CF-1 mice were given T₃ in their drinking water to suppress endogenous TSH. T₃, 3.0 μg/mL, given to mice for a period of 4 days decreased plasma total T₄ (TT₄) level to less than 10% of the nonsuppressed (control) level. Various doses of exogenous pituitary and recombinant TSH preparations were injected intraperitoneally and blood samples were obtained from the orbital sinus 6 h later. The TT₄ level, measured by radioimmunoassay, served as the assay end-point. The assay required injection of approximately 3.0 μg of pituitary human TSH (phTSH), 1.0 Ltg recombinant human TSH (rhTSH), 0.2 μg bovine TSH (bTSH), and 0.1 μg rat TSH (rTSH) to attain half-maximal response. The maximal level of TT₄ after TSH stimulation was similar to that observed in normal, nonsuppressed mice. The procedure developed is relatively easy to perform, economical, and, unlike earlier TSH bioassays, does not require the use of radionuclides. This bioassay showed acceptable sensitivity and reliability in structure-function studies of pituitary TSH from different species as well as rhTSH.