Spontaneous DNA breakage is predicted to be a frequent, inevitable consequence of DNA replication and is thought to underlie much of the genomic change that fuels cancer and evolution. Despite its importance, there has been little direct measurement of the amounts, types, sources and fates of spontaneous DNA lesions in living cells. We present a direct, sensitive flow cytometric assay in single living Escherichia coli cells for DNA lesions capable of inducing the SOS DNA damage response, and we report its use in quantification of spontaneous DNA double-strand breaks (DSBs). We report efficient detection of single chromosomal DSBs and rates of spontaneous breakage approximately 20- to 100-fold lower than predicted. In addition, we implicate DNA replication in the origin of spontaneous DSBs with the finding of fewer spontaneous DSBs in a mutant with altered DNA polymerase III. The data imply that spontaneous DSBs induce genomic changes and instability 20-100 times more potently than previously appreciated. Finally, FACS demonstrated two main cell fates after spontaneous DNA damage: viability with or without resumption of proliferation.