Cellular stimulation results in phosphorylation of the tumor suppressor p53 on multiple residues, though the functional relevance is not always clear. It is noteworthy that the serine (S) 315 residue is unique, as it has been suggested to be phosphorylated not only by genotoxic signals, but also during cell-cycle progression and by endoplasmic-reticulum stress. However, in vitro data have been conflicting as phosphorylation at this site was shown to both positively and negatively regulate p53 functions. We have thus generated knock-in mice expressing an unphosphorylable S312 (equivalent to human S315), by substitution with an alanine (A) residue, to clarify the conflicting observations and to evaluate its functional relevance in vivo. Born at Mendelian ratios, the p53(S312A/S312A) mice show no anomalies during development and adulthood. p53 activation, stability, localization and ability to induce apoptosis, cell-cycle arrest and prevent centrosome amplification are not compromised in p53(S312A/S312A) cells. p53(S312A/S312A) mice are unable to rescue mdm2(-/-) lethality, and tumorigenesis--both spontaneous and irradiation/oncogene-induced--is not accentuated. Taken together, the results show that the S312 phosphorylation site is not in itself necessary for efficient p53 function, and advocates the possibility that it is neither relevant in the mouse context nor important for p53 functions in vivo.