Eukaryotic cells respond to ionizing radiation exposure with cell cycle arrest. However, little is known about the signaling mechanisms responsible for this effect. The present work has asked whether ionizing radiation exposure is associated with changes in phosphorylation of proteins in HL-60 myeloid leukemia cells. The results demonstrate increased tyrosine phosphorylation of a M(r) 34,000 substrate. This effect was detectable at 1 to 10 min after irradiation and was induced by doses of 50 to 500 cGy. Immunoprecipitation studies further suggest that this substrate is the serine/threonine p34cdc2 protein kinase. Since p34cdc2 is required for entry into mitosis, these findings support the posttranslational modification of a cell cycle regulatory protein in the response to ionizing radiation.