Background/Aims: To test the activity of novel hydroxyvitamin D3 analogs (20(OH)D3, 20,23(OH)2D and 1,20(OH) 2D3) on normal and malignant melanocytes in comparison to 1,25(OH)2D3. Materials and Methods: Human epidermal melanocytes and human and hamster melanoma cells were used to measure effects on proliferation and colony formation in monolayer and soft agar. Cell morphology and melanogenesis were also analyzed. QPCR was used to measure gene expression. Results: Novel secosteroids inhibited proliferation and colony formation by melanoma cells in a similar fashion to 1,25(OH)2D3, having no effect on melanogenesis. These effects were accompanied by ligand-induced translocation of VDR to the nucleus. In normal melanocytes 1α- hydroxyderivatives (1,25(OH)2D3 and 1,20(OH) 2D3) had stronger anti-proliferative effects than 20(OH)D3 and 20,23(OH)2D3, and inhibited dendrite formation. The cells tested expressed genes encoding VDR and enzymes that activate or inactivate vitamin D3. Conclusion: Novel secosteroids show potent anti-melanoma activity in vitro with 20(OH)D 3 and 20,23(OH)2D3 being excellent candidates for pre-clinical testing.