HL-60 promyelocytic leukemia cells differentiated to eosinophils and eosinophilic precursors when cultured under mildly alkaline conditions (pH 7.6-7.8) for 7 d without refeeding. New cytoplasmic granules appeared blue in the least mature cells and red in the most mature cells when stained with Wright-Giemsa. The granules also stained with Luxol-fast-blue, a characteristic of eosinophil granules. Furthermore, most cells contained the eosinophil major basic protein (MBP); the Charcot-Leyden Crystal (CLC) protein (lysophospholipase), eosinophil peroxidase, acid phosphatase, and arylsulfatase were also detected in a portion of these cells. The eosinophil major basic protein was found in a high proportion of undifferentiated cells, and thus may be constituitively produced. By examining finely banded chromosomes, translocation break points were demonstrated at q22 on one chromosome 16 and at q23 on the other homologue; abnormalities in this region of the long arm of 16 are a characteristic finding in the recently described syndrome of acute myelomonocytic leukemia (AMMoL) with abnormal bone marrow eosinophils. In common with the bone marrow eosinophils in these patients, the HL-60 eosinophil granules contained chloroacetate esterase and periodic-acid Schiff (PAS) reactive material; crystalloid inclusions were rare. Therefore, the HL-60 cell line appears to be an in vitro model for eosinophilopoiesis and may be specially suited for the study of the abnormal eosinophils seen in certain malignant conditions.