Primary suspension cultures from serially transplanted mouse pituitary thyrotropic tumors were regulated by physiological levels of thyroid hormones. TSH [thyrotropin] release was linear for up to 48 h in control cultures and was inhibited progressively after 10, 24 and 48 h in cultures exposed to triiodothyronine (T3), 4.0 nM. TSH release was inhibited up to 55% of control and glucose consumption was stimulated up to 2.6-fold in a dose-dependent fashion by T3 between 0.2 and 4.0 nM. A biphasic dose-dependent relationship for T3 and thyroxine (T4), and TSH production was demonstrated in 2 series of cultures. TSH production was stimulated progressively by T3 up to 0.1 nM and T4 up to 5 nM. At higher T3 and T4 levels, TSH production was progressively inhibited. Half-maximal inhibition occurred at total medium concentrations of 0.2 nM T3 and 15 nM T4 or free hormone levels of 8 .times. 10-12 M and 14 .times. 10-11 M, respectively. High affinity, low capacity nuclear binding sites were demonstrated for T3 and T4. The apparent equilibrium dissociation constants were 0.16 nM for T3 and 1.7 nM for T4 in serum-free medium. There were approximately 14,000 molecules of T3 or T4 bound per cell nucleus at saturation. These data suggest that T3 is approximately 15-fold more potent than T4 in regulating TSH production and cellular metabolism in these cells, and these effects may be initiated by interaction with nuclear binding sites. Within the physiological range, low levels of thyroid hormones stimulate and higher levels inhibit TSH production.