Culture supernatants from established human fibroblast cell lines (FCM) and from phytahemagglutinin-stimulated peripheral blood leukocytes (LCM) were compared with respect to their stimulatory effects on the colony formation by human marrow leukocyte precursors in vitro using the methylcellulose culture system. The cultures were performed using specimens from children with a variety of disorders. LCM stimulation consistently produced a significantly higher proportion of macrophage colonies than did FCM stimulation, whereas FCM stimulation resulted in a significantly higher number of number of neutrophil colonies. Based on the sequential examination of colony numbers in 35 FCM-and 11 LCM- stimulated cultures, the colonies in LCM-stimulated cultures (predominantly macrophage colonies) survived much longer than those in FCM-stimulated cultures (predominantly neutrophil colonies). Cell fractionation experiments by velocity sedimentation on four different specimens revealed that the majority of FCM-responsive cells were large and formed pure neutrophil colonies, while the majority of LCM- responsive cells were smaller and formed either pure macrophage or mixed neutrophil-macrophage colonies. These observations suggest that human colony-forming cells consist of at least two distinct cell populations different in (1) cell size, (2) response to two different conditioned media, LCM and FCM, and (3) type of colonies each population forms.