Previously, we have shown that primary cultures of murine calvarial cells produce both granulocyte macrophage (GM) and granulocyte (G) colony stimulating factor (CSF). Because of the heterogeneity of cell types in these cultures the osseous origin of these cytokines was not certain. Thus a non-transformed rat clonal osteoblastic cell population CRP 10/30 and the immortalized cell line IRC10/30-myc1 derived from it, which both express the osteoblastic phenotype, were now investigated. Both produced hemopoietic growth activity after treatment with recombinant murine tumor necrosis factor alpha. This activity eluted from diethylaminoethyl Sephacel at 0.2-0.3 M NaCl, and migrated on Sephacryl S-200 at a mol wt of around 30 k, as described for murine GM- and G-CSF. On a Phenyl Sepharose CL-4B column, it was separated into two peaks appearing at position where GM (peak I) and G-CSF (peak II) are expected to be eluted. Antisera against GM-CSF inhibited the activity of peak I. In the colony assay in semisolid medium, peak I induced colonies of the GM-type and peak II of the G-type. These data indicate that cloned osteoblasts produce GM- and G-CSF. Through CSF production, osteoblasts might regulate osteoclast formation, influence hemopoiesis and/or participate in local inflammatory reactions of bone.