This work is aimed at exploring regulatory mechanisms involved in normal differentiation and malignant transformation using the B16 melanoma model system involving melanotic and amelanotic malignant clones and amelanotic nonmalignant clones derived from the melanotic clone after growth and maintenance in the presence of 1 microgram/ml of 5-bromodeoxyuridine (BrdUrd). the BrdUrd clone can immunize syngeneic mice against the parental tumor. By using hyperimmune mouse antisera to the BrdUrd-grown clone and to irradiated parental malignant cells we are comparing glycoproteins and proteins between the 2 cell types by immunoprecipitation, immuodiffusion, immunofluorescence and cytotoxicity. Peritoneal exudate cells from immune animals can neutralize the tumorigenic ability of the malignant cells when coinjected into syngeneic mice. Macrophage:T cell cooperation appears to be involved. Delineation of the T cell type is underway. Peritoneal exudate cells from mice immunized with heat-killed BCG also neutralize malignant cells. The effector cells appear to be macrophages. We have shown that correlation between plasminogen activator (PA) activity and tumorigenicity and between inhibition of PA activity and tumorigenicity need not exist. We are examining the mechanism by which 5-azacytidine and other regulators of differentiated function operate. These approaches should lead to further understanding of regulatory mechanisms in malignancy, immuogenicity and melanogenesis.