Melanoma-associated antigens were isolated from human melanoma cells in long-term tissue culture and from the spent culture fluid of these cells propagated in chemically defined, serum-free media. The 3 M KCl extracts from such cells and their concentrated spent culture media elicited specific delayed cutaneous hypersensitivity reactions in patients with malignant melanoma but not in patients with other neoplasms. HLA antigens present in these extracts could be specifically removed by ultracentrifugation in KBr at a density of 1.23 g/ml. Purification of melanoma-associated antigens was achieved by this step, followed by ion-exchange chromatography and preparative isoelectric focusing on Pevikon C870. Another approach is described for the isolation of carcionembryonic antigens from metastatic lesions with an approximately 70% yield utilizing the least denaturing procedures, which avoid lyophilization and involve essentially 0.9% NaCl solution extraction, specific adsorption, elution from concanavalin A Sepharose, and subsequent gel-exclusion chromatography on Ultrogel AcA 22. For effective isolation of carcinoembryonic antigens freely shed from cultured cells derived from a primary colon tumor, a system was devised based on the use of Amicon hollow fiber culture units, in which cultured tumor cells were introduced in the extracapiliary spaces of such a unit. The extracapillary fluid, containing carcinoembryonic antigens but no fetal calf serum components, is removed and further purified by affinity chromatography.