The involvement of colony-stimulating activity (CSA) in murine long- term bone marrow cultures (LTBMC) was studied using bilayer agar cultures. The supernatants of LTBMC were removed, a layer of dense agar was spread over the cells adherent to the bottom of the flask, and fresh myeloid cells were plated as source of CFU-C in an upper agar layer. Large numbers of granulocytic and macrophagic colonies developed regularly when target cells were plated over adherent cells of nonrecharged and greater than 12 wk old LTBMC that were hematopoietically inactive (i.e., producing a low number of nonadherent cells). The removal of adherent cells from the myeloid cells used as source of CFU-C did not decrease the number of colonies. This suggests that adherent cells of LTBMC release CSA that is directly active on CFU- C. This CSA was no longer detectable over adherent layers of hematopoietically active LTBMC. A close inverse relationship was demonstrated between the number of nonadherent cells harvested before the assay and the level of CSA. No inhibitor for CSA was demonstrated in the supernatant of hematopoietically active cultures. Murine exogenous CSA incubated over the adherent layer host its activity within 24 hr, whereas in the same conditions human CSA retained its activity. These data demonstrate the production of CSA by the adherent layer of LTBMC and strongly suggest its specific in situ consumption by differentiating myeloid cells.