Every day cells are exposed to different stresses that may cause DNA damage. UV radiation and certain anti-cancer drugs mainly cause single-strand DNA breaks, leading to the activation of the ATR pathway and consequently of the tumour suppressor p53. Actually, p53 is a central node in the DNA damage response pathways. Studies on this protein have mainly been made at the population level using classic biochemistry approaches. In recent years, single-cell microscopy analysis, using plasmid-based expression systems, have revealed that p53 shows a series of nuclear translocations in response to double-strand DNA damage, a behaviour that can be masked at the population level. In the present work, a BAC transgenesis system containing the p53 gene tagged to dsRedXP was improved and transiently transfected into MCF7 cells. By using this system it was possible to see that, in contrast with what has been proposed in the past, cells mainly displayed multiple p53 translocations (peaks) in response to UV damage. However, there was an increase in the number of cells showing a single, albeit wider, p53 peak as the UV dose increased, suggesting a longer p53 occupancy within the nucleus. As UV dose increased, the cumulative levels of p53 in the nucleus also appeared to increase, as possibly a higher level of repair was needed. Interestingly, with this work it was possible to see that even though the time of appearance of the first p53 nuclear translocations following UV stress was very variable, the timing of consecutive p53-dsRedXP nuclear translocations as well as nuclear import and export rates of this protein were very stable, independently of level of stress. Through population-based qRT-PCR assays it was also possible to see that the activation of p21, Mdm2 and Wip1, all p53 transcriptional targets, appeared to occur within the same time as the average first p53 nuclear translocation following UV damage. Cells also appeared to present a basal p53 response against damage, which was similar to the response seen in cells exposed to a low dose of UV. Different cancer cell lines were also shown to present a different wiring of the p53 network, leading to different profiles of mRNA response. Overall, with this work it was possible to show that the choice of transgenesis system to study the single-cell behaviour of p53 is of utmost importance. p53 shows a very heterogeneous behaviour in cells exposed to UV stress, even within a defined dose, thus showing that its response to damage and activity is not only dependent on the type and level of stress but possibly on the location of the damage and other factors inherent to each cell and its local environment.