GCT, a human monocyte-like cell line, releases biochemically distinct colony-stimulating activities (CSAs) for mouse and human marrows. These appear to be periodate-sensitive proteins with critical disulfide bonds. One, of MW 145,000 daltons, stimulates mouse macrophagic colony growth and is related to a 30,000-dalton MW molecule that also stimulates mouse growth. A 30,000-dalton CSA for human marrow can be separated from the 30,000-dalton mouse CSA by isoelectric focusing and gradient polyacrylamide gel electrophoresis. This distinction agrees with the previous finding of differential neutralization with anti-human urinary CSF antibody. The 30,000-dalton CSA stimulate neutrophil, neutrophil-monocyte, and eosinophil colony growth in human marrow but only neutrophil and neutrophil-monocyte colonies in the mouse. Subcellular fractionation of GCT cells indicates that there are pools of preformed CSA primarily associated with the cell cytosol that have similar apparent MW to their secreted counterparts.