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 cells 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.