Murine bone marrow and adherence-separated spleen cells cultured on hydrophobic, gas-permeable Teflon foils (petriperm dishes) can be shown to synthesize and secrete erythropoietin (Epo) and colony-stimulating activity (CSA) simultaneously into the surrounding medium. The Epo activity in the supernatants of primary cultures as measured by the fetal liver erythroid colony-forming technique, from adherent and nonadherent spleen cells, increases over the first 7 days in culture, followed by a plateau until 14 days. Use of the macrophage-specific cytotoxic agent, crystalline silica, as a tool to release residual Epo contained in these cells produces a similar time-Epo activity curve to that found in the primary supernatants. This, together with functional and morphological examination of the cells, indicates that macrophages are responsible for this activity. The total Epo activity released from adherent and nonadherent spleen cells at plateau levels was estimated to be 25 mU/ml culture/day. Weekly subcultivation of bone marrow and adherence-separated spleen cells initiated from primary cultures demonstrated a massive increase in both Epo activity and CSA above that obtained for the primary cultures. Subcultivation could be continued for at least 6 wk. These results, together with the reversible inhibition of Epo and CSA production by cycloheximide, demonstrate that these molecules are synthesized by the macrophage. The evidence supports the hypothesis that the macrophage is involved not only in extrarenal Epo production, but also in the possible short-range regulation of hemopoiesis.