53BP1 and NFBD1/MDC1 are recruited rapidly to sites of DNA double-strand breaks (DSBs), where they are hypothesized to function downstream of the ataxia-telangiectasia mutated (ATM) checkpoint kinase as "mediators" of DNA DSB signaling. To test this hypothesis, we suppressed 53BP1 and NFBD1/MDC1 expression by small interference RNA and monitored ATM autophosphorylation at Ser(1981) as a marker for ATM activation. Suppression of NFBD1/MDC1 led to decreased ATM activation and phosphorylation of ATM substrates. This phenotype was identical to that observed in cells with defective Nbs1 function and is consistent with recent observations identifying NFBD1/MDC1 as a component of the Mre11-Rad50-Nbs1 protein complex. In cells with wild-type Nbs1, suppression of 53BP1 expression had no effect on ATM activation but was associated with increased recruitment of NFBD1/MDC1 and Nbs1 to sites of DNA breaks, suggesting that decreased 53BP1 function might be compensated for by increased NFBD1/MDC1 and Nbs1 activity. Indeed, in cells with mutant Nbs1, suppression of 53BP1 led to decreased ATM activation and phosphorylation of ATM substrates. We conclude that DNA DSBs activate ATM through at least two independent pathways involving 53BP1 and NFBD1/MDC1-Nbs1, respectively.