The effects of clinically relevant ethanol concentrations on myeloid differentiation in the HL-60 cell promyelocytic leukemia line have been studied. The exposure of noninduced stem cells to 60 mM ethanol results in an increase in G1 cells, but there is no increase in superoxide production or expression of the Mo1 antigen. When HL-60 cells are induced to differentiate along the myeloid line with dimethylsulfoxide (DMSO) or retinoic acid (RA), there is a shift to smaller cell size, an increase in G1 cells and acquisition of the ability to produce superoxide as reported previously by several authors. When ethanol is present during differentiation, there are further increases in G1 cells, and increases in the percentage of cells which produce superoxide and express Mo1, and decreases in mean cell size and total growth during the incubation period. Regrowth experiments after periods of differentiation indicate that the increased G1 arrest seen in the presence of ethanol represents terminal commitment if inducer is present, but in the absence of inducer the increased G1 percentage is readily reversible. Examination of RNA content by flow cytometry reveals a decrease in both the peak and mean G1 RNA content during DMSO or RA induced differentiation. These decreases are accentuated by the presence of ethanol, resulting in a higher G1A/G1B ratio than in nonexposed cells. These findings indicate that ethanol enhances G1 growth arrest in HL-60 cells exposed to myeloid inducers. Partial differentiation occurs during this process, resulting in terminally arrested cells, some of which have undergone fewer postinduction cell divisions than normal and may not be fully competent.