HL-60 cells can be induced to differentiate into mature granulocytic cells in a continuous exposure to all-trans retinoic acid (RA) for at least 96 h. In this study, RA was removed after being exposed to HL-60 cells for 12-96h. Then the cells were reincubated in a fresh medium without drug for additional 48-144h and their differentiation markers such as NBT reduction, morphological changes and cytokinetic characteristics were studied. The results demonstrated that once exposed continuously to 1.0 mumol/L RA for at least 48h, the HL-60 cells would proceed to terminal differentiation: about 60-90 percent of the cells became NBT positive, 40-80 percent of cells exhibited decreased nucleus/cytoplasm ratio and marked reduction in the number of nucleoli and displayed matured granulocytic morphology. Cytokinetic studies demonstrated 48-85 percent of cells arrested in G1 phase, which is typical for differentiated cells. However, if the cells were exposed to RA for less than 48h, they would not show differentiation phenotype. These results indicate that 48h exposure is critical for HL-60 cell differentiation induced by RA, and the cell differentiation induced by RA is irreversible and RA-independent.