A screen of a library of 2,000 drugs and natural products in murine melanocytes identified 10 tricyclic antidepressants (TCAs) as compounds that potently decreased intracellular melanin content. The rank order of potency of these compounds for decreasing melanin content was different than their relative potencies as antidepressants. These compounds had no effect on either the level or the enzymatic activity of cellular tyrosinase (Tyr). Increased presence of both Tyr and melanin in the culture media was observed in treated melanocytes. Immunofluorescence localization revealed that these compounds decreased intracellular melanin content by disrupting the intracellular trafficking of Tyr gene family proteins. In treated melanocytes, Tyr, Tyr-related protein 1, and dopachrome tautomerase accumulated in enlarged granules distributed throughout the cytoplasm. Colocalization of Tyr with lysosome-associated membrane protein 1 was observed within many of these granules. Partial colocalization of Tyr with the Hermansky-Pudlak syndrome 1 gene product observed in control melanocytes was abolished by TCA treatment. Our results show that these compounds decreased intracellular melanin content by altering the trafficking of Tyr gene family proteins and inducing abnormal secretion of Tyr. Results from our screening have implications for the design of products for skin lightening and treatment of hyperpigmentation.