Human choroidal melanocytes were isolated from eye bank tissue and cultured in vitro with 10 ng/ml cholera toxin and 10 ng/ml phorbol myristate acetate. These growth factors induced rapid proliferation of spindle-type melanocytes. Similar results were obtained using 10 mM putrescine. Phenotypic analysis of these cells by immunoperoxidase staining with anti-HLA-DR monoclonal antibodies revealed 40-70% positive cells. HLA-DR positive melanocytes were isolated and purified from the HLA-DR negative population by rosetting with protein-A coupled sheep erythrocytes followed by differential centrifugation. Ultrastructural analysis of choroidal melanocytes revealed evidence of premelanosomes and melanosomes. Choroidal melanocytes in tissue culture are important experimental controls for the study of the cell biology of choroidal melanoma.