Human histone H2AX is rapidly phosphorylated on serine 139 in response to DNA double-strand breaks and plays a crucial role in tethering the factors involved in DNA repair and damage signaling. Replication stress caused by hydroxyurea or UV also initiates H2AX phosphorylation in S-phase cells, although UV-induced H2AX phosphorylation in non-cycling cells has recently been observed. Here we study the UV-induced H2AX phosphorylation in human primary fibroblasts under growth-arrested conditions. This reaction absolutely depends on nucleotide excision repair (NER) and is mechanistically distinct from the replication stress-induced phosphorylation. The treatment of cytosine-β-D-arabinofuranoside strikingly enhances the NER-dependent H2AX phosphorylation and induces the accumulation of replication protein A (RPA) and ATR-interacting protein (ATRIP) at locally UV-damaged subnuclear regions. Consistently, the phosphorylation appears to be mainly mediated by ataxia-telangiectasia mutated and Rad3-related (ATR), although Chk1 (Ser345) is not phosphorylated by the activated ATR. The cellular levels of DNA polymerases δ and ϵ and proliferating cell nuclear antigen are markedly reduced in quiescent cells. We propose a model that perturbed gap-filling synthesis following dual incision in NER generates single-strand DNA gaps and hence initiates H2AX phosphorylation by ATR with the aid of RPA and ATRIP.