We have established two-human cell lines which elaborate colony-stimulating activity (CSA) for at least four species: man, mouse, rabbit, and dog. One GCT, was isolated from a lung metastasis of a fibrous histiocytoma; the other RC4, from a monocyte-enriched fraction of normal blood. Medium conditioned by either GCT or RC4 cells was more potent in stimulating human marrow growth in vitro than was monocyte-conditioned medium or human leukocyte feeder layers. Fractionation of cell line-conditioned medium by Sephacryl S-200 chromatography indicated that the maximum activity of the CSA for human marrow cells is eluted within the range of 30,000-40,000 daltons. These cell lines provide a continuous source of large quantities of conditioned medium for purification of CSA. Moreover, the invariable growth-supporting activity for all species tested and the high potency of cell-line CSA facilitates studies of its elaboration and biologic effects.