The first step during the genome duplication is the initiation of DNA replication, i.e., the recognition of the DNA replication origins (ORIs) by the appropriate protein complexes, the loading of the DNA helicase (origin licensing) and the first DNA unwinding (origin firing). Towards a better comprehension of this process in human cells, we initially aimed to investigate the molecular mechanisms of ORI recognition, focusing on the aspect of sequence specificity. We found that a fraction of human ORIs contained a conserved motif, correspondent to the binding site for the AP-1 transcription factor and exhibited a reduced firing efficiency upon its deletion. In parallel, we studied the process of origin licensing and more precisely the period during the G1 phase when it occurs. We found that ORIs are licensed throughout the G1 phase, as licensing inhibition at any G1 timeframe impaired the entry into S phase and significantly reduced the origin firing efficiency. We are currently aiming to define whether different steps of origin licensing occur in early vs late G1 phase.