Four monocytoid cell lines, JOSK-I, -S, -M, and -K, were newly established successfully from peripheral blood of two cases of acute monocytic leukemia and one case each of acute myelomonocytic leukemia and chronic myelogenous leukemia in myelomonocytic blast crisis. In order to establish permanent cell lines, cultures of leukemic blasts were initiated in 96-well microtiter plates. Each cell line grew in a suspension culture with a doubling time of 24-32 h and has been serially maintained for over 20 mo. Each line had immature monocytic properties as judged from the results of cytological, immunochemical, and functional analyses. The cells showed a positive reaction for alpha-naphthyl butyrate esterase which was completely inhibited by sodium fluoride and exhibited immature monocytic features on electron microscopic observation. They also had surface markers specific for the monocyte-macrophage lineage. Chromosome analyses showed that each line had a variety of marker chromosomes; furthermore, these established lines exhibited high potentialities involving morphological and functional differentiation into more mature monocytic cells when induced by several chemical inducers. We also found that two of the established cell lines produced much interleukin 1 activity without any stimuli. These new lines might be valuable for studying the regulation of monocyte-macrophage differentiation and host defense mechanisms.