Investigations with mouse melanoma B16/C3 cell cultures have suggested that imidazole or a derivative thereof can facilitate expression of the tyrosinase (EC 1.14.18.1) structural gene. The induction of tyrosinase expression by imidazole was inhibited by T3 about 4-fold. When T3 (10 nM) was present for 19 h in proliferating B16/C3 cultures, basal activity of this enzyme was inhibited by approximately 60%. Neither T3 nor imidazole directly affected tyrosinase enzymatic activity in broken cell preparations. Addition of T3 to imidazole-induced cultures rapidly decreased tyrosinase expression (within 30 min) which remained repressed for at least 4 h before recovering. Recovery of tyrosinase activity could be blocked by readdition of hormone. The hormone effect was detectable at 1 nM and was maximal at 10 nM. Removal of supplemental T3 from the medium rapidly reversed the repression of tyrosinase activity. The biologically inactive analog rT3 (10 nM) failed to inhibit basal enzyme activity or alter the imidazole effect on tyrosinase expression. The experimental results with protein and RNA inhibitors indicate that the T3 response is independent of destabilizing the putative transcript for tyrosinase or altering the posttranslational events responsible for its synthesis.