Modulation of TSH [thyroid stimulating hormone] release from mouse thyrotropic tumor cells was studied. T3 [Triiodothyronine] (1 nM) inhibited basal TSH release, while 6 nM T3 blocked TRH[thyroliberin]-induced TSH release. Prior exposure of cells to actinomycin or cycloheximide prevented T3 from suppressing basal and TRH-induced TSH release. The TSH-suppressive activity from T3-treated cells was extracted and exposure of untreated thyrotropic cells to this material suppressed TSH release. T3 suppression of TSH may be mediated by formation of an inhibitory protein in thyrotropic cells.