The transcription factor NF-kappaB has critical functions in biologic responses to genotoxic stimuli. Activation of NF-kappaB in response to DNA double strand break (DSB) inducers can be mediated by ATM (ataxia telangiectasia mutated)-dependent phosphorylation of NEMO (NF-kappaB essential modulator). Here, we show that the replication stress inducers hydroxyurea (HU) and aphidicolin also activate this ATM-dependent signalling pathway. We further show that ATR (ATM- and Rad3-related) interacts with NEMO but surprisingly does not cause NEMO phosphorylation. Consequently, ATR represses NF-kappaB activation induced by replication stress. Reduction or increase of ATR expression by RNA interference or overexpression increased or reduced ATM-NEMO association and NF-kappaB activation induced by HU. Apoptosis gene expression and chromatin immunoprecipitation analyses indicated that HU and the DSB inducer etoposide caused complex patterns of NF-kappaB-dependent pro- and antiapoptotic gene expression with the overall outcome for the former being pro-apoptotic, whereas the latter antiapoptotic. Thus, replication stress and DSB inducers activate NF-kappaB through a conserved pathway with opposite biologic outcomes, and ATR antagonizes ATM function at least in part by competing for NEMO association.