The long-term suspension growth of normal, immature myeloid cells from fresh human cord blood was recently reported and required cells separated on supplemented discontinuous Percoll gradients, growth in media containing hydrocortisone and vitamins D3, and gentle, continuous agitation (13). When normal adult bone marrow (six donors) or blood from Epstein-Barr virus (EBV)-seropositive donors (nine donors) was used as a source of fresh human leukocytes, only short-term proliferation of myeloid cells was achieved with the same techniques. However, when leukocytes prepared from EBV seronegative normal adult peripheral blood were used, pure populations of monocytes and macrophages that replicate slowly in liquid suspension culture for greater than 5 mo were repeatedly obtained from three independent donors. These cultures consists of several morphologically distinguishable monocytic cell types, including an approximately 20% adherent macrophage population. The monocytic nature of these cultures was confirmed by cytochemical, immunological, and functional criteria. These monocytes retain a normal chromosome pattern and can be induced to differentiate to phagocytic cells by treatment with tetradecanylphorbal acetate. Eventually, the cultures terminate as nonreplicating mature macrophages. These liquid suspension cultures should be a valuable resource for morphological, biochemical, and functional studies of developing monocyte-macrophages and their interaction with other cell types in normal and various pathological situations.