The p53 protein contains several functional domains necessary for inducing cell cycle arrest and apoptosis. The C-terminal basic domain within residues 364-393 and the proline-rich domain within residues 64-91 are required for apoptotic activity. In addition, activation domain 2 within residues 43-63 is necessary for apoptotic activity when the N-terminal activation domain 1 within residues 1-42 is deleted (DeltaAD1) or mutated (AD1(-)). Here we have discovered that an activation domain 2 mutation at residues 53-54 (AD2(-)) abrogates the apoptotic activity but has no significant effect on cell cycle arrest. We have also found that p53-(DeltaAD2), which lacks activation domain 2, is inert in inducing apoptosis. p53-(AD2(-)DeltaBD), which is defective in activation domain 2 and lacks the C-terminal basic domain, p53-(DeltaAD2DeltaBD), which lacks both activation domain 2 and the C-terminal basic domain, and p53-(DeltaPRDDeltaBD), which lacks both the proline-rich domain and the C-terminal basic domain, are also inert in inducing apoptosis. All four mutants are still capable of inducing cell cycle arrest, albeit to a lesser extent than wild-type p53. Interestingly, we have found that deletion of the N-terminal activation domain 1 alleviates the requirement of the C-terminal basic domain for apoptotic activity. Thus, we have generated a small but potent p53-(DeltaAD1DeltaBD) molecule. Furthermore, we have determined that at least two of the three domains (activation domain 1, activation domain 2, and the proline-rich domain), are required for inducing cell cycle arrest. Taken together, our results suggest that activation domain 2 and the proline-rich domain form an activation domain for inducing pro-apoptotic genes or inhibiting anti-apoptotic genes. The C-terminal basic domain is required for maintaining this activation domain competent for transactivation or transrepression.