Cultures of the promyelocytic leukemia cell line HL-60 usually contain considerable numbers of spontaneously differentiating cells and are asynchronous in terms of cell-cycle phases. Counterflow centrifugal elutriation studies have been conducted to obtain a homogeneous cell population with regard to cell-cycle phases and stage of differentiation. Despite their small volume and probably because of their high buoyant density, differentiated cells are elutriated predominantly at higher flow rates. Accordingly, G1 cells elutriated at low flow rates are substantially free from spontaneously differentiating cells. By optimizing the technique, a population with approx. 90% G1 cells and less than 1% spontaneously differentiating cells was obtained. The yield in the fractions chosen was 5.1% of all cells recovered from elutriation. In culture, a cell population of this purity maintains a synchronous cell cycle for more than 2 days. This allows an exact determination of the time after induction when the first signs of differentiation occur. The presence of 1 microM retinoic acid (RA) causes the first significant increase of NBT-positive cells between the 24th and 27th h of culture.