We used intrachromosomal substrates to directly monitor the effect of the cell cycle on the efficiency and the accuracy of nonhomologous end joining (NHEJ) in mammalian cells. We show that both KU and KU-independent (KU-alt) pathways are efficient when maintaining cells in G1/S, in G2/M or during dynamic progression through S phase. In addition, the accuracy of NHEJ is barely altered when the cells are blocked in G1/S or in G2/M. However, progression through S phase increases the frequency of deletions, which is a hallmark of the KU-alt pathway. Moreover, we show that the intermediates that are generated by the KU-dependent end joining of non-fully complementary ends, and which contain mismatches, nicks or gap intermediates, are less accurately processed when the cells progress through S phase. In conclusion, both KU and KU-alt processes are active throughout the cell cycle, but the repair is more error prone during S phase, both by increasing the mutagenic KU-alt pathway and decreasing the accuracy of the repair of the intermediates generated by the KU-dependent pathway.