Caffeine inhibits cell cycle checkpoints, sensitizes cells to ionizing radiation-induced cell killing and inhibits the protein kinase activity of two cell cycle checkpoint regulators, Ataxia-Telangiectasia mutated (ATM) and ATM- and Rad3-related (ATR). In contrast, caffeine has been reported to have little effect on the protein kinase activity of the DNA-dependent protein kinase (DNA-PK), which is essential for the repair of DNA double-strand breaks. Previously, we reported that DNA-PK phosphorylates Thr21 of the 32 kDa subunit of replication protein A (RPA32) in response to camptothecin. In this report we demonstrate that the camptothecin-induced phosphorylation of RPA32 on Thr21 is inhibited by 2 mM caffeine. In addition, we show that caffeine inhibits immunoprecipitated and purified DNA-PK, as well as DNA-PK in cell extracts, with an IC50 of 0.2-0.6 mM. Caffeine inhibited DNA-PK activity through a mixed non-competitive mechanism with respect to ATP. In contrast, 10-fold higher concentrations of caffeine were required to inhibit DNA-PK autophosphorylation in vitro and caffeine failed to inhibit DNA-PKcs dependent double-strand break repair in vivo. These data suggest that while DNA-PK does not appear to be the target of caffeine-induced radiosensitization, caffeine cannot be used to differentiate between ATM, ATR and DNA- PK-dependent substrate phosphorylation in vivo.