Ataxia-telangiectasia mutated (ATM) encodes a nuclear serine/threonine protein kinase whose activity is increased in cells exposed to low doses of ionizing radiation (IR). Here we examine ATM kinase activation in cells exposed to either 32 P- or 33 P-orthophosphate under conditions typically employed in metabolic labelling experiments. We calculate that the absorbed dose of IR delivered to a 5cm×5cm monolayer of cells incubated in 2ml media containing 1mCi of the high-energy (1.70MeV) β-particle emitter 32 P-orthophosphate for 30min is ∼1Gy IR. The absorbed dose of IR following an otherwise identical exposure to the low-energy (0.24MeV) β-particle emitter 33 P-orthophosphate is ∼0.18Gy IR. We show that low-energy β-particles emitted by 33 P induce a greater number of ionizing radiation-induced foci (IRIF) and greater ATM kinase signaling than energetic β-particles emitted by 32 P. Hence, we demonstrate that it is inappropriate to use 33 P-orthophosphate as a negative control for 32 P-orthophosphate in experiments investigating DNA damage responses to DNA double-strand breaks (DSBs). Significantly, we show that ATM accumulates in the chromatin fraction when ATM kinase activity is inhibited during exposure to either radionuclide. Finally, we also show that chromosome aberrations accumulate in cells when ATM kinase activity is inhibited during exposure to ∼0.36Gy β-particles emitted by 33 P. We therefore propose that direct cellular exposure to 33 P-orthophosphate is an excellent means to induce and label the IR-induced, ATM kinase-dependent phosphoproteome.