This report shows that hydroxyl radical, generated by a Fenton reaction involving adenosine 5'-diphosphate/Fe2+ complex (5-15 ㄍM) and H2O2 (2 ㄍM), induced differentiation of HL-60 cells in a dose- and time-dependent manner. This is evidenced by the increases in 12-O-tetradecanoylphorbol 13-acetate- and fMLP-stimulated superoxide production capability. The cells exposed to hydroxyl radical for defined periods (24∼96 hr) continued to differentiate even after the hydroxyl radical generating system had been removed. The differentiated cells displayed fMLP-stimulated calcium mobilization and increased expression of myeloid-specific antigen CD11b and CD14. The extent of the differentiation was markedly reduced by desferrioxamine (100 ㄍM), dimethylthiourea (5 mM), N,N'-diphenyl-1,4-phenylenediamine (2 ㄍM), and N-acetyl-L-cysteine (5 mM). The induction of differentiation by hydroxyl radical was enhanced by 3-isobutyl-1-methylxanthine (200 ㄍM) and Ro-20-1724 (8 ㄍM), and inhibited by dipyridamole (2 ㄍM). These results suggest that hydroxyl radicals may induce commitment of HL-60 cells to differentiate into more mature cells of myelomonocytic lineage through specific signal-transduction pathway that is modulated by phosphodiesterase inhibitors.