A monocytic cell line, termed Mono Mac, was established from peripheral blood of a patient with monoblastic leukemia. Two clones, designated Mono Mac I and Mono Mac 6, were isolated and both were assigned to the monocyte lineage on the basis of morphological, cytochemical and immunological criteria. Most importantly, the clones express NaF‐sensitive non‐specific‐esterase, produce reactive oxygen and stain with MAb My4. Mono Mac 6, in addition, constitutively exhibits phagocytosis of antibody‐coated erythrocytes in 80% of the cells and reacts with a panel of MAbs that are specific for mature monocytes, i.e., M42, LeuM3, 63D3, Mo2 and UCHMI. By contrast, the monoblastic cell lines U937 and THP‐I are negative for all these markers. Only expression of My4 could be detected after differentiation induced by interferon‐γ (IFN‐γ). Similar treatment of Mono Mac I, however, resulted in staining with all the monocyte‐specific MAbs mentioned above, while IFN‐γ treatment of Mono Mac 6 enhanced antigen expression. In addition, the cells showed an increased frequency of multinucleated cells with a rise from 4.8% to 21.9%. Mono Mac 6 appears to be the only one of the cell lines studied to constitutively express phenotypic and functional features of mature monocytes.