The activity of the tumor suppressor p53 is induced in response to DNA-damaging agents such as UV and gamma radiation. Phosphorylation is one of the key regulatory steps for activating p53 function. Recent reports have shown that p53 is phosphorylated at both serines 15 and 392 in response to UV radiation. Phosphorylation at serine 15 prevents the binding of HDM2, a negative regulator of p53. Phosphorylation at serine 392 induces the DNA-binding function of p53. We examined the requirement for phosphorylation at both serines and show that both these modifications occur on the same molecule of p53. In vitro assays demonstrate that phosphorylation at either one of these sites is not sufficient to yield an active p53. Phosphorylation by DNA-PK, that modifies serines 15 and 37, inhibits HDM2 binding to p53 but does not induce the DNA-binding activity of p53. Phosphorylation at serine 392, on the other hand, stimulates the DNA-binding ability of p53 but does not make it immune to binding and inhibition by HDM2. Thus, our results demonstrate that multiple sites need to be modified to yield a functional p53.