To study the regulation of macrophage (m phi) heterogeneity at bone marrow level, we developed a liquid culture system in which bone marrow-derived soft-agar colonies were expanded in the presence of colony-stimulating factor-1 (CSF-1). Several cloned m phi precursor cells were established as CSF-1-dependent cell lines, and were analyzed for morphological, phenotypic and functional characteristics. The continuously proliferating cell lines expressed both immature and mature m phi markers. Only minor differences between the established cell lines were detected. Thus, our results show that during long-term culture CSF-1-responsive cloned m phi precursor cells develop along identical pathways, giving rise to progeny with comparable phenotype and function in vitro.