Micropthalmia-associated transcription factor (MITF) is a lineage restricted basic helix-loop-helix leucine zipper transcription factor that is essential for melanocyte development, function and survival. 15% of human melanomas have MITF gene amplification (1). In addition, a vast majority of melanomas are dependent upon MITF for survival. We set out to identify small molecule inhibitors of MITF activity that would allow for better molecular characterization of MITF's role in melanoma. Using an MITF-dependent melanoma cell line, SK-MEL-5, in a cell-based luminescence assay, we measured the promoter activity of a MITF target gene, melastatin (TRPM-1), in a high throughput screen (HTS). 331,578 compounds from the NIH MLPCN compound library were screened. Of these, 3,206 compounds were active (a hit rate of 0.96%). A chloronaphthoquinone (CID 1716436/SID 22416871) was identified in the primary HTS as an inhibitor of TRPM-1 promoter activity. It had potent activity upon retesting in the primary assay, as did several closely related analogs. Structure activity relationship (SAR) studies were performed to improve potency and to minimize deleterious properties. These efforts generated a probe (CID 12387471/ML329) with improved chemical properties and selectivity. In particular, ML329 was not prone to nucleophilic glutathione addition, whereas the initial hit underwent adduct formation. ML329 was tested in two MITF-dependent melanoma cell viability assays, SK-MEL-5 and MALME-3M plus a MITF-independent cell line, A375. ML329 showed specific activity against the MITF-dependent cells, primary melanocytes but no effect on the viability in A375 cells. ML329 reduced the expression of multiple MITF target genes, including pigment-related genes and the cell cycle regulator CDK2. As a tool compound, ML329 will be useful in elucidating the role of MITF in melanocyte lineage development and in melanoma disease progression.