At any moment during S phase, regions of genomic DNA are in various stages of replication (i.e., initiation, chain elongation, and termination). These stages may be differentially inhibited after treatment with various carcinogens that damage DNA such as UV. We used visualization of active replication units in combed DNA fibers, in combination with quantitative analyses of the size distributions of nascent DNA, to evaluate the role of S-checkpoint proteins in UV-induced inhibition of DNA replication. When HeLa cells were exposed to a low fluence (1 J/m(2)) of 254 nm UV light (UVC), new initiation events were severely inhibited (5-6-fold reduction). A larger fluence of UVC (10 J/m(2)) resulted in stronger inhibition of the overall rate of DNA synthesis without decreasing further the frequency of replicon initiation events. Incubation of HeLa cells with caffeine and knockdown of ATR or Chk1 kinases reversed the UVC-induced inhibition of initiation of new replicons. These findings illustrate the concordance of data derived from different experimental approaches, thus strengthening the evidence that the activation of the intra-S checkpoint by UVC is dependent on the ATR and Chk1 kinases.