BCG was fractionated into a delipidated mycobacterial cell fraction (DMC) and lipid by exhaustive chloroform-methanol extraction. The effects of these fractions were tested on mouse spleen cells, nonadherent spleen cells (lymphocytes), thymus cells, and adherent spleen cells (macrophages) in vitro and were compared with effects of the whole bacilli and a methanol-extraction residue (MER). Tritiated thymidine incorporation into spleen cells, purified spleen lymphocytes, and thymus cells was measured as an indicator of activity on these cells; lymphocyte-activating factor (LAF) production was used to measure activation of macrophages. DMC and MER were at least equivalent to, and often exceeded, whole BCG in their stimulation of spleen cells and spleen lymphocytes. DMC was a poor thymic mitogen in contrast to MER, which was as strong as BCG in this regard. Lipid was far less effective a mitogen for all cells tested, and failed to augment the effectiveness of DMC on thymus cells when both were present in the incubation mixture. LAF production was significantly increased by whole BCG (18-fold above controls), whereas each fraction increased production threefold to sixfold. These in vitro results seemed to reflect the known in vivo activity of BCG and its components and suggest further antitumor applications.