This study was aimed at investigating whether cells of CFU-C derived colonies could form secondary colonies. Bone marrow cultures of volumes of agar medium between 25 microliter and 75 microliter contained in glass capillaries were stimulated with mouse lung-conditioned medium (MLCM) containing granulocyte/macrophage colony-stimulating factor (GM-CSF). Agar gels with colonies of up to greater than or equal to 20 were blown out into identical culture medium, completely dispersed on a whirl-mix to single cell suspensions, and used for establishing secondary agar cultures. In these secondary cultures considerable numbers of secondary granulocytic, mixed granulocytic/macrophage and macrophage colonies as well as numerous clusters arose. In contrast, when single colonies were recultured, only few secondary cell aggregates were formed. When primary cultures containing up to greater than or equal to 20 cell aggregates were used for serial reculture at intermittent intervals of 3 and 4 days, a 2-7-fold increase of colony-forming cells was found in tertiary cultures as was monitored by 7 day colony counts. And by use of different kinds of CSF-containing media, an over 4-fold increase of secondary over primary colonies was obtained with bovine lung-conditioned medium (BLCM) in primary and L-cell-conditioned medium (LCCM) in secondary 7 day cultures. Primary capillary cultures were found to be devoid of CFU-S. Also, setting up bone marrow cultures in petri dishes and stimulating with MLCM, growth of primary as well as secondary colonies was obtained. The results indicate some self-renewal potential of CFU-C in vitro.