The p53 gene encodes several isoforms that can interfere with stress responses by modulating p53 wild-type (wt) function. Recently, a C-terminally truncated splice variant, p53beta, has been implicated in the regulation of p53-dependent apoptosis, whereas the function of similarly spliced p53gamma was not investigated before. Therefore, we studied the impact of these isoforms on the function of endogenous p53wt and compared it with that of exogenously expressed p53wt. We demonstrate that despite an efficient nuclear expression of all isoforms, only p53wt modulates apoptosis induction. Furthermore, only p53wt assembles into a transcription-competent oligomeric complex or translocates to mitochondria upon stress induction. Both C-terminally truncated isoforms fail to modulate the apoptotic function of p53wt because they are unable to associate with p53wt and hence do not bind to p53 DNA recognition sequences. Consistently, the dominant-negative function of transactivation-deficient Delta133p53 is completely lost in the Delta133p53beta variant. Intriguingly, the alternatively spliced C-terminus protects p53beta and p53gamma not only from MDM2-mediated proteasomal degradation, but strongly impairs their binding to this negative regulator. Thus, our data demonstrate the necessity of the regularly spliced C-terminal tail for multiple layers of p53 regulation and function.