Freshly explanted human myeloma cells formed colonies of monoclonal plasma cells in soft agar in the presence of medium conditioned by the adherent spleen cells of mineral oil-primed BALB/c mice. The medium showed peak activity at a dilution of 1:4. 2-mercaptoethanol or monothioglycerol was necessary for colony formation. Other thiols tested were ineffective in promoting colony growth. Colony-forming cells adhered to nylon wool, but not glass beads or plastic dishes. The presence of E-rosetting cells was not required for myeloma colony formation. Antibody prepared against a human myeloma cell line, RPMI 8226, reduced colony formation. These studies demonstrate the usefulness of this bioassay for determining functional properties of the myeloma colony-forming cell.