Mammalian cells respond to ionizing radiation (IR) with cell cycle arrest, activation of DNA repair, and induction of early response genes. The present work has examined the involvement of Src-like protein-tyrosine kinases in the response of irradiated HL-60 myeloid leukemia cells. The results demonstrate little if any effect of IR on p59fyn, p56lck, and pp60c-src activity. In contrast, HL-60 cells responded to x-ray exposure with activation of p56/p53lyn. At a dose of 200 centigrays, induction of p56/p53lyn activity was detectable at 15 min. Doses as low as 50 centigrays were effective in activating p56/p53lyn. H2O2 and the scavenger N-acetylcysteine had no detectable effect on p56/p53lyn activation, while the protein-tyrosine kinase inhibitors, herbimycin and genistein, blocked induction by IR. The results also demonstrate that incubation of a glutathione S-transferase-Lyn fusion protein with lysates of irradiated HL-60 cells is associated with binding of the cell cycle regulatory protein, p34cdc2. The interaction of p56/p53lyn and p34cdc was confirmed in similar experiments with a glutathione S-transferase-Cdc2 fusion protein. Moreover, coimmunoprecipitation studies demonstrate the selective binding of activated p56/p53lyn to p34cdc2 in irradiated cells. These findings indicate that IR activates p56/p53lyn in HL-60 cells and that this tyrosine kinase may contribute to the regulation of p34cdc2.