UV irradiation induces histone variant H2AX phosphorylated on serine 139 (γH2AX) foci and high levels of pan-nuclear γH2AX staining without foci, but the significance of this finding is still uncertain. We examined the formation of γH2AX and 53BP1 that coincide at sites of double-strand breaks (DSBs) after ionizing radiation. We compared UV irradiation and treatment with etoposide, an agent that causes DSBs during DNA replication. We found that during DNA replication, UV irradiation induced at least three classes of γH2AX response: a minority of γH2AX foci colocalizing with 53BP1 foci that represent DSBs at replication sites, a majority of γH2AX foci that did not colocalize with 53BP1 foci, and cells with high levels of pan-nuclear γH2AX without foci of either γH2AX or 53BP1. Ataxia-telangiectasia mutated kinase and JNK mediated the UV-induced pan-nuclear γH2Ax, which preceded and paralleled UV-induced S phase apoptosis. These high levels of pan-nuclear γH2AX were further increased by loss of the bypass polymerase Pol η and inhibition of ataxia-telangiectasia and Rad3-related, but the levels required the presence of the damage-binding proteins of excision repair xeroderma pigmentosum complementation group A and C proteins. DSBs, therefore, represent a small variable fraction of UV-induced γH2AX foci dependent on repair capacity, and they are not detected within high levels of pan-nuclear γH2AX, a preapoptotic signal associated with ATM- and JNK-dependent apoptosis during replication. The formation of γH2AX foci after treatment with DNA-damaging agents cannot, therefore, be used as a direct measure of DSBs without independent corroborating evidence.