The p53 tumour suppressor governs cell fate by differential transactivation of a spectrum of target genes. To further understand how p53 discriminates between target promoters, we have for the first time used in vitro compartmentalization (IVC) to evolve variants with greater affinity for the distal p53 response element in the promoter of the p21 gene involved in cell-cycle arrest, and for the low affinity BS1 response element of the pro-apoptotic PUMA gene. These variants have mutations in the L1 loop of the p53 DNA binding domain and in the N-terminal proline-rich domain. The in vitro binding phenotype of these variants extends to both increased transactivation of promoters containing the response elements in reporter gene studies and increased up-regulation of endogenous p21 as compared to wild-type p53. One variant was co-selected for increased binding to both response elements yet displayed increased apoptotic function. This result supports the notion that prediction of phenotypic outcome based on transcriptional activation of individual genes is confounded by the networked complexity of the p53 response.